MDM2-mediated P53 Degradation Research Articles

A new PICTure of nucleolar stress

Cell growth demands new protein synthesis, which requires nucleolar ribosomal functions. Ribosome biogenesis consumes a large proportion of the cell's resources and energy, and so is tightly regulated through an intricate signaling network to guarantee fidelity. Thus, events that impair ribosome biogenesis cause nucleolar stress. In response to this stress, several nucleolar ribosomal proteins (RPs) translocate to the nucleoplasm and bind to MDM2. MDM2-mediated ubiquitination and degradation of the tumor suppressor p53 is then blocked, resulting in p53 accumulation and the induction of p53-dependent cell cycle arrest and apoptosis. Nucleolar stress is therefore a quality control surveillance mechanism that monitors the synthesis and assembly of the rRNA and protein components of ribosomes. Although nucleolar stress signaling pathways have been extensively analyzed, critical questions remain about their regulatory mechanisms. For example, how do RPs translocate from the nucleolus to the nucleoplasm to exert their functions, and do these p53-regulating RPs influence the prognosis of human cancer patients? Our laboratory recently identified the nucleolar protein PICT1 as a novel regulator of nucleolar stress. PICT1 sequesters the ribosomal protein RPL11 in the nucleolus, preventing it from binding to MDM2. MDM2 is then free to degrade p53, favoring tumor cell growth. Accordingly, the level of PICT1 in a tumor is becoming a useful prognostic marker for human cancers. This review summarizes the evidence that links nucleolar stress to tumorigenesis, and casts PICT1 as an oncogenic player in human cancer biology.

Multifaceted functions of Siva-1: more than an Indian God of Destruction

Siva-1, as a p53-inducible gene, has been shown to induce extensive apoptosis in a number of different cell lines. Recent evidence suggests that Siva-1 functions as a part of the auto-regulatory feedback loop that restrains p53 through facilitating Mdm2-mediated p53 degradation. Also, Siva-1 plays an important role in suppressing tumor metastasis. Here we review the current understanding of Siva-1-mediated apoptotic signaling pathway. We also add comments on the p53-Siva-1 feedback loop, the novel function of Siva-1 in suppressing tumor metastasis, and their potential implications.

Impairment of p53 acetylation by EWS-Fli1 chimeric protein in Ewing Family Tumors

The chromosomal translocation t(11;22)(q24;q12) yields the EWS-Fli1 fusion gene, which contributes to the development of Ewing Family Tumors (EFTs). Previous studies have shown the ability of EWS-Fli1 chimeric protein to silence p53 activity. Here we demonstrate that the introduction of EWS-Fli1 significantly inhibited p300-mediated acetylation of p53 at Lys-382 and depletion of EWS-Fli1 protein by small interfering RNAs (siRNA) in EFTs cells facilitated it in response to DNA damage. Furthermore, the deacetylation of p53 by EWS-Fli1 suppressed its transcriptional activity and enhanced mdm2-mediated p53 degradation. On the other hand, immunoprecipitation study shows that N-terminal region of EWS-Fli1 associated with histone deacetylase 1 (HDAC1) to forms a complex with p53.Knockdown of HDAC1, but not HDAC2 or HDAC3 protein restored the expression of p53 Lys-382 in EFTs cells. Overexpression of HDAC1 also significantly inhibited p53 transcriptional activity. Pharmacologic inhibitor of HDAC, trichostatin A (TSA) promoted p53–p300 interaction and recruitment of p53 Lys-382 to promoter regions of its target genes p21 and Puma, consequently inducing apoptosis and stabilizing the acetylation of p53 at Lys-382 together with the upregulation of p21 and Puma, which were impaired in EFTs cells after the knockdown of p53 expression. Our data indicate EWS-Fli1 might deacetylate p53 to inhibit its transcriptional function and protein stability via the recruitment of HDAC1. These results might elucidate a novel molecular mechanism about the abrogation of p53 pathway by EWS-Fli1 in EFTs pathogenesis.

A crucial role for bone morphogenetic protein-Smad1 signalling in the DNA damage response

DNA damage and the elicited cellular response underlie the etiology of tumorigenesis and ageing. Yet, how this response integrates inputs from cells' environmental cues remains underexplored. Here we report that the BMP-Smad1 pathway, which is essential for embryonic development and tissue homeostasis, has an important role in the DNA damage response and oncogenesis. On genotoxic stress, Atm phosphorylates BMPs-activated Smad1 in the nucleus on S239, which disrupts Smad1 interaction with protein phosphatase PPM1A, leading to enhanced activation and upregulation of Smad1. Smad1 then interacts with p53 and inhibits Mdm2-mediated p53 ubiquitination and degradation to regulate cell proliferation and survival. Enhanced Smad1 S239 phosphorylation, and Smad1 mutations causing S239 substitution were detected in oesophageal and gastric cancer samples, respectively. These findings suggest that BMP-Smad1 signalling participates in the DNA damage response via the Atm-p53 pathway, thus providing a molecular mechanism whereby BMP-Smad1 loss-of-function leads to tumorigenesis, for example, juvenile polyposis and Cowden syndromes.

Abrogation of Gli3 expression suppresses the growth of colon cancer cells via activation of p53

p53, the major human tumor suppressor, appears to be related to sonic hedgehog (Shh)–Gli-mediated tumorigenesis. However, the role of p53 in tumor progression by the Shh–Gli signaling pathway is poorly understood. Herein we investigated the critical regulation of Gli3–p53 in tumorigenesis of colon cancer cells and the molecular mechanisms underlying these effects. RT-PCR analysis indicated that the mRNA level of Shh and Gli3 in colon tumor tissues was significantly higher than corresponding normal tissues (P<0.001). The inhibition of Gli3 by treatment with Gli3 siRNA resulted in a clear decrease in cell proliferation and enhanced the level of expression of p53 proteins compared to treatment with control siRNA. The half-life of p53 was dramatically increased by treatment with Gli3 siRNA. In addition, treatment with MG132 blocked MDM2-mediated p53 ubiquitination and degradation, and led to accumulation of p53 in Gli3 siRNA-overexpressing cells. Importantly, ectopic expression of p53 siRNA reduced the ability of Gli3 siRNA to suppress proliferation of those cells compared with the cells treated with Gli3 siRNA alone. Moreover, Gli3 siRNA sensitized colon cancer cells to treatment with anti-cancer agents (5-FU and bevacizumab). Taken together, our studies demonstrate that loss of Gli3 signaling leads to disruption of the MDM2–p53 interaction and strongly potentiate p53-dependent cell growth inhibition in colon cancer cells, indicating a basis for the rational use of Gli3 antagonists as a novel treatment option for colon cancer.

Reciprocal repression between P53 and TCTP

Screening for genes that reprogram cancer cells for the tumor reversion switch identified TCTP (encoding translationally controlled tumor protein) as a crucial regulator of apoptosis. Here we report a negative feedback loop between P53 and TCTP. TCTP promotes P53 degradation by competing with NUMB for binding to P53-MDM2-containing complexes. TCTP inhibits MDM2 auto-ubiquitination and promotes MDM2-mediated ubiquitination and degradation of P53. Notably, Tctp haploinsufficient mice are sensitized to P53-dependent apoptosis. In addition, P53 directly represses TCTP transcription. In 508 breast cancers, high-TCTP status associates with poorly differentiated, aggressive G3-grade tumors, predicting poor prognosis (P < 0.0005). Tctp knockdown in primary mammary tumor cells from ErbB2 transgenic mice results in increased P53 expression and a decreased number of stem-like cancer cells. The pharmacological compounds sertraline and thioridazine increase the amount of P53 by neutralizing TCTP's action on the MDM2-P53 axis. This study links TCTP and P53 in a previously unidentified regulatory circuitry that may underlie the relevance of TCTP in cancer.

Crosstalk between MYCN and MDM2-p53 signal pathways regulates tumor cell growth and apoptosis in neuroblastoma

Previous studies show that the MYCN and MDM2-p53 signal pathways are mutually regulated: MYCN stimulates MDM2 and p53 transcription, whereas MDM2 stabilizes MYCN mRNA and induces its translation. Herein, we report that the interaction between MDM2 and MYCN plays a critical role in MYCN-amplified neuroblastoma tumor cell growth and survival. Distinct from the known role that MDM2 has in regulating tumor promotion in non-MYCN-amplified neuroblastoma, in which MDM2 inhibits p53, we found that MDM2 stimulated tumor growth in MYCN-amplified neuroblastoma in a p53-independent manner. In MYCN-amplified neuroblastoma cells, enforced expression of MDM2 further enhanced MYCN expression, yet no p53 inhibition was observed by MDM2 due to upregulation of MYCN that stimulated p53 transcription. Similarly, p53 expression remained unchanged in MDM2-silenced MYCN-amplified neuroblastoma cells because MDM2 inhibition resulted in a downregulation of MYCN that decreased p53 transcription, although the MDM2-mediated degradation of p53 was reduced. Also, we found that the enforced overexpression of MDM2, or conversely, the inhibition of overexpressed endogenous MDM2, led to either a remarkable increase or decrease in tumor growth, respectively, in MYCN-amplified neuroblastoma (even though no p53 function was involved). These results suggest that p53 that is reciprocally regulated by MDM2 and MYCN is dispensable for suppression of MYCN-amplified neuroblastoma, and that the direct interaction between MDM2 and MYCN may contribute significantly to MYCN-amplified neuroblastoma growth and disease progression.

Abstract 3087: Regulation of p53 stability and activity by ribosomal protein S27a

Abstract Ribosomal proteins (RPs) play a critical role in tightly coordinating p53-dependent cell cycle checkpoint with ribosomal biogenesis. A number of RPs, including L5, L11, L23, L26, and S7, specifically bind to MDM2 and inhibit MDM2-mediated p53 ubiquitination and degradation, leading to p53 activation, in response to nucleolar stress triggered by the perturbation of ribosomal biogenesis. Here, we report S27a, a ribosomal protein from small subunit of ribosome and synthesized as an ubiquitin C-terminal extension protein (CEP80), as a novel MDM2 regulator. S27a interacts with MDM2 at the central acidic domain of MDM2 and suppresses MDM2-mediated p53 ubiquitination, leading to p53 activation and cell cycle arrest. Knockdown of endogenous S27a significantly attenuated p53 activation by treatment with a low dose (5 nM) of actinomycin D (Act D), which specifically inhibits RNA polymerase I activity leading to inhibition of rRNA synthesis and perturbation of ribosomal biogenesis, suggesting that S27a is a non-redundant MDM2 regulator in response to nucleolar stress. Interestingly, we also show that MDM2 ubiquitinates S27a, leading to protesomal degradation of S27a, in response to Act D treatment, suggesting that MDM2, in turn, controls S27a levels in response to nucleolar stress. Taken together, our results reveal that S27a regulates p53 activity in response to nucleolar stress via a novel and dynamic interplay between S27a and MDM2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3087. doi:10.1158/1538-7445.AM2011-3087

P21WAF1 is component of a positive feedback loop that maintains the p53 transcriptional program

The regulation of p53 activity through the MDM2 negative feedback loop is driven in part by an extrinsic ATM-pulse that maintains p53 oscillations in response to DNA damage. We report here that the p53 pathway has evolved an intrinsic positive feedback loop that is maintained by the p53-inducible gene product p21WAF1. p21-null cancer cells have defects in p53 protein turnover, reductions in MDM2-mediated degradation of p53, and reduced DNA damage-induced ubiquitination of p53. TLR3-IRF1 or ATM-dependent signaling to p53 is defective in p21-null cells and complementation of the p21 gene in p21-null cancer cells restores the p53 transcriptional response. The mechanism of p53 inactivity in p21-null cells is linked to a p53 protein equilibrium shift from chromatin into cytosolic fractions and complementation of the p21 gene into p21-null cells restores the nuclear localization of p53. A loss of p53 transcriptional function in murine B-cells heterozygous or homozygous null for p21 highlights a p21-gene dosage effect that maintains the full p53 transcriptional response. ATM inhibition results in nuclear exclusion of p53 highlighting a positive genetic interaction between ATM and p21. P21 protein oscillates in undamaged proliferating cells, and reductions of p21 protein using siRNA eliminate the DNA damage-induced p53 pulse. The p53 transcription program has evolved a negative feedback loop maintained by MDM2 that is counteracted by a positive feedback loop maintained by ATM-p21 the balance of which controls the specific activity of p53 as a transcription factor.

The balance between rRNA and ribosomal protein synthesis up- and downregulates the tumour suppressor p53 in mammalian cells

Data on the relationship between ribosome biogenesis and p53 function indicate that the tumour suppressor can be activated by either nucleolar disruption or ribosomal protein defects. However, there is increasing evidence that the induction of p53 does not always require these severe cellular changes, and data are still lacking on a possible role of ribosome biogenesis in the downregulation of p53. Here, we studied the effect of the up- and downregulation of the rRNA transcription rate on p53 induction in mammalian cells. We found that a downregulation of rRNA synthesis, induced by silencing the POLR1A gene coding for the RNA polymerase I catalytic subunit, stabilised p53 without altering the nucleolar integrity in human cancer cells. p53 stabilisation was due to the inactivation of the MDM2-mediated p53 degradation by the binding of ribosomal proteins no longer used for ribosome building. p53 stabilisation did not occur when rRNA synthesis downregulation was associated with a contemporary reduction of protein synthesis. Furthermore, we demonstrated that in three different experimental models characterised by an upregulation of rRNA synthesis, cancer cells treated with insulin or exposed to the insulin-like growth factor 1, rat liver stimulated by cortisol and regenerating rat liver after partial hepatectomy, the p53 protein level was reduced due to a lowered ribosomal protein availability for MDM2 binding. It is worth noting that the upregulation of rRNA synthesis was responsible for a decreased p53-mediated response to cytotoxic stresses. These findings demonstrated that the balance between rRNA and ribosomal protein synthesis controls the function of p53 in mammalian cells, that p53 can be induced without the occurrence of severe changes of the cellular components controlling ribosome biogenesis, and that conditions characterised by an upregulated rRNA synthesis are associated with a reduced p53 response.

Human T-lymphotropic Virus Type 1 p30 Interacts with REGγ and Modulates ATM (Ataxia Telangiectasia Mutated) to Promote Cell Survival

Human T-lymphotropic virus type 1 (HTLV-1) is a causative agent of adult T cell leukemia/lymphoma and a variety of inflammatory disorders. HTLV-1 encodes a nuclear localizing protein, p30, that selectively alters viral and cellular gene expression, activates G(2)-M cell cycle checkpoints, and is essential for viral spread. Here, we used immunoprecipitation and affinity pulldown of ectopically expressed p30 coupled with mass spectrometry to identify cellular binding partners of p30. Our data indicate that p30 specifically binds to cellular ATM (ataxia telangiectasia mutated) and REGγ (a nuclear 20 S proteasome activator). Under conditions of genotoxic stress, p30 expression was associated with reduced levels of ATM and increased cell survival. Knockdown or overexpression of REGγ paralleled p30 expression, suggesting an unexpected enhancement of p30 expression in the presence of REGγ. Finally, size exclusion chromatography revealed the presence of p30 in a high molecular mass complex along with ATM and REGγ. On the basis of our findings, we propose that HTLV-1 p30 interacts with ATM and REGγ to increase viral spread by facilitating cell survival.

Ubiquitin-dependent and Ubiquitin-independent Control of Subunit Stoichiometry in the SWI/SNF Complex

The mammalian SWI/SNF chromatin remodeling complex is a key player in multiple chromatin transactions. Core subunits of this complex, including the ATPase, Brg-1, and various Brg-1-associated factors (BAFs), work in concert to maintain a functional remodeling complex. This intra-complex regulation is supervised by protein-protein interactions, as stoichiometric levels of BAF proteins are maintained by proteasomal degradation. We show that the mechanism of BAF155-mediated stabilization of BAF57 involves blocking its ubiquitination by preventing interaction with TRIP12, an E3 ubiquitin ligase. Consequently, as opposed to complexed BAF57, whose principal lysines are unavailable for ubiquitination, uncomplexed BAF57 can be freely ubiquitinated and degraded by the proteasome. Additionally, a BAF57 mutant, which contains no lysine residues, was found to retain its ability to be stabilized by interaction with BAF155, suggesting that in addition to the ubiquitin-dependent mechanism of BAF57 degradation, there exists a ubiquitin-independent mechanism that may involve the direct interaction of BAF57 with the proteasome. We propose that this regulatory mechanism exists to ensure functional fidelity of the complex and prevent the accumulation of uncomplexed proteins, which may disrupt the normal activity of the complex.

Exploring a minimal two-component p53 model

The tumor suppressor p53 coordinates many attributes of cellular processes via interlocked feedback loops. To understand the biological implications of feedback loops in a p53 system, a two-component model which encompasses essential feedback loops was constructed and further explored. Diverse bifurcation properties, such as bistability and oscillation, emerge by manipulating the feedback strength. The p53-mediated MDM2 induction dictates the bifurcation patterns. We first identified irradiation dichotomy in p53 models and further proposed that bistability and oscillation can behave in a coordinated manner. Further sensitivity analysis revealed that p53 basal production and MDM2-mediated p53 degradation, which are central to cellular control, are most sensitive processes. Also, we identified that the much more significant variations in amplitude of p53 pulses observed in experiments can be derived from overall amplitude parameter sensitivity. The combined approach with bifurcation analysis, stochastic simulation and sampling-based sensitivity analysis not only gives crucial insights into the dynamics of the p53 system, but also creates a fertile ground for understanding the regulatory patterns of other biological networks.

Mitochondrial HEP27 Is a c-Myb Target Gene That Inhibits Mdm2 and Stabilizes p53

The ever-expanding knowledge of the role of p53 in cellular metabolism, apoptosis, and cell cycle control has led to increasing interest in defining the stress response pathways that regulate Mdm2. In an effort to identify novel Mdm2 binding partners, we performed a large-scale immunoprecipitation of Mdm2 in the osteosarcoma U2OS cell line. One significant binding protein identified was Hep27, a member of the short-chain alcohol dehydrogenase/reductase (SDR) family of enzymes. Here, we demonstrate that the Hep27 preprotein contains an N-terminal mitochondrial targeting signal that is cleaved following mitochondrial import, resulting in mitochondrial matrix accumulation of mature Hep27. A fraction of the mitochondrial Hep27 translocates to the nucleus, where it binds to Mdm2 in the central domain, resulting in the attenuation of Mdm2-mediated p53 degradation. In addition, Hep27 is regulated at the transcriptional level by the proto-oncogene c-Myb and is required for c-Myb-induced p53 stabilization. Breast cancer gene expression analysis correlated estrogen receptor (ER) status with Hep27 expression and p53 function, providing a potential in vivo link between estrogen receptor signaling and p53 activity. Our data demonstrate a unique c-Myb-Hep27-Mdm2-p53 mitochondria-to-nucleus signaling pathway that may have functional significance for ER-positive breast cancers.

Cholesterol Perturbation in Mice Results in p53 Degradation and Axonal Pathology through p38 MAPK and Mdm2 Activation

Perturbation of lipid metabolism, especially of cholesterol homeostasis, can be catastrophic to mammalian brain, as it has the highest level of cholesterol in the body. This notion is best illustrated by the severe progressive neurodegeneration in Niemann-Pick Type C (NPC) disease, one of the lysosomal storage diseases, caused by mutations in the NPC1 or NPC2 gene. In this study, we found that growth cone collapse induced by genetic or pharmacological disruption of cholesterol egress from late endosomes/lysosomes was directly related to a decrease in axonal and growth cone levels of the phosphorylated form of the tumor suppressor factor p53. Cholesterol perturbation-induced growth cone collapse and decrease in phosphorylated p53 were reduced by inhibition of p38 mitogen-activated protein kinase (MAPK) and murine double minute (Mdm2) E3 ligase. Growth cone collapse induced by genetic (npc1−/−) or pharmacological modification of cholesterol metabolism was Rho kinase (ROCK)-dependent and associated with increased RhoA protein synthesis; both processes were significantly reduced by P38 MAPK or Mdm2 inhibition. Finally, in vivo ROCK inhibition significantly increased phosphorylated p53 levels and neurofilaments in axons, and axonal bundle size in npc1−/− mice. These results indicate that NPC-related and cholesterol perturbation-induced axonal pathology is associated with an abnormal signaling pathway consisting in p38 MAPK activation leading to Mdm2-mediated p53 degradation, followed by ROCK activation. These results also suggest new targets for pharmacological treatment of NPC disease and other diseases associated with disruption of cholesterol metabolism.

Harmonic Oscillations in Homeostatic Controllers: Dynamics of the p53 Regulatory System

Homeostatic mechanisms are essential for the protection and adaptation of organisms in a changing and challenging environment. Previously, we have described molecular mechanisms that lead to robust homeostasis/adaptation under inflow or outflow perturbations. Here we report that harmonic oscillations occur in models of such homeostatic controllers and that a close relationship exists between the control of the p53/Mdm2 system and that of a homeostatic inflow controller. This homeostatic control model of the p53 system provides an explanation why large fluctuations in the amplitude of p53/Mdm2 oscillations may arise as part of the homeostatic regulation of p53 by Mdm2 under DNA-damaging conditions. In the presence of DNA damage p53 is upregulated, but is subject to a tight control by Mdm2 and other factors to avoid a premature apoptotic response of the cell at low DNA damage levels. One of the regulatory steps is the Mdm2-mediated degradation of p53 by the proteasome. Oscillations in the p53/Mdm2 system are considered to be part of a mechanism by which a cell decides between cell cycle arrest/DNA repair and apoptosis. In the homeostatic inflow control model, harmonic oscillations in p53/Mdm2 levels arise when the binding strength of p53 to degradation complexes increases. Due to the harmonic character of the oscillations rapid fluctuating noise can lead, as experimentally observed, to large variations in the amplitude of the oscillation but not in their period, a behavior which has been difficult to simulate by deterministic limit-cycle models. In conclusion, the oscillatory response of homeostatic controllers may provide new insights into the origin and role of oscillations observed in homeostatically controlled molecular networks.

Inhibition of the p53–MDM2 interaction by adenovirus delivery of ribosomal protein L23 stabilizes p53 and induces cell cycle arrest and apoptosis in gastric cancer

Inhibiting MDM2 activity in tumors that express wild-type (wt) p53 but have high levels of MDM2 protein has been considered an attractive anticancer strategy for many years. Previous studies revealed that human ribosomal protein L23 (RPL23) inhibited MDM2-mediated p53 degradation and thus induced p53 levels as well as its activity, suggesting that it might be a candidate for use as a gene therapy for cancer. In the present study, we evaluated whether targeting this pathway could be of therapeutic value against human gastric carcinoma. Gastric cancer cell lines carrying wt or mutant p53 gene were infected with adenovirus expressing RPL23 (Ad-RPL23). Cell growth assay, flow cytometry assay and morphology were used to observe the effects of Ad-RPL23 infection on tumor cells, and further, the effect of Ad-RPL23 treatment on tumor growth in vivo was investigated. In vitro, adenovirus-mediated RPL23 gene transfer stabilized wt p53 by inhibiting its degradation, and thus resulted in G(1)-S cell cycle arrest and/or apoptosis of human gastric cancer MKN45 and AGS cells carrying wt p53 gene. Adenovirus-mediated RPL23 gene transfer also inhibited the growth of MKN45 tumors in subcutaneous mouse models. The data obtained in the present study suggest that, through the inhibition of the p53-MDM2 interaction, adenovirus delivery of RPL23 can inhibit the proliferation of gastric cancer cells harboring wt p53 in vitro and in vivo. Exogenous RPL23-induced wt p53 stabilization and activation may be a novel therapeutic approach for patients with gastric cancer.

Oncogenic RAS Enables DNA Damage- and p53-Dependent Differentiation of Acute Myeloid Leukemia Cells in Response to Chemotherapy.

Abstract 5040 IntroductionAcute myeloid leukemia (AML) is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. High-dose cytarabine is used as the standard consolidation chemotherapy. Oncogenic RAS mutations are frequently observed in AML, and patients with RAS mutations benefit most from high-dose cytarabine as postremission therapy (Neubauer et al., J Clin Oncol 2008). The molecular reason for this phenomenon is not well understood. MethodsWe used bone marrow cells expressing a conditional MLL-ENL-ER oncogene to investigate the interaction of oncogenic RAS and chemotherapeutic agents. In addition, we used primary human inversion 16 positive AML samples with or without oncogenic RAS mutations to corroborate our findings. ResultsThere was no difference in cell cycle kinetics, apoptosis or cellular senescence in MLL-ENL cell expressing control vector vs. MLL-ENL cells expressing oncogenic RAS in response to cytarabine treatment. However, we observed an increased activation of DNA damage checkpoints in MLL-ENL-cells expressing oncogenic RAS after incubation with chemotherapeutic agents. This resulted in an Atm/r as well as p53-dependent genetic program causing dramatically reduction of clonogenicity in MLL-ENL cells expressing oncogenic RAS due to induction of myeloid differentiation. Co-expression of dominant – negative p53 completely abolished this differentiation resulting in the rescue of the clonogic potential. Activation of p53 as a result of inhibition of Mdm2-mediated degradation of p53 further enhances this myeloid differentiation. Of note, in primary AML cases, oncogenic RAS also was associated with a more differentiated phenotype. ConclusionsThe data can explain the beneficial effects observed in AML patients with oncogenic RAS mutations treated with high dosages of cytarabine and suggest that induction of p53-dependent differentiation, e.g. by interfering with Mdm2-mediated degradation, may be a rational approach to increase cure rate in response to chemotherapy since stronger acitvation of p53 by oncogenic RAS and chemotherapeutic agents cause the differentiation of hematopoietic stem cells to more differentiated progenitors. The data also support the notion that the therapeutic success of cytotoxic drugs may depend on their ability to promote the differentiation of tumor-initiating cells. DisclosuresNo relevant conflicts of interest to declare.

Secondary Resistance to Lenalidomide in Del(5q) MDS Is Associated with CDC25C & PP2A Overexpression.

Abstract 292 Background:Allelic deficiency for the RPS14 gene impairs differentiation and survival of erythroid progenitors in del(5q) MDS (Nature 2008; 451:335). Nucleolar stress arising from disruption of ribosome assembly fosters MDM2 sequestration by free ribosome components resulting in p53 stabilization and erythroid hypoplasia (Nat Cell Biol 2009; 11:501). We recently reported that reduced gene dosage of the lenalidomide (LEN) inhibitable, haplodeficient phosphatases CDC25C and PP2Acα is a key determinant of drug sensitivity in del(5q) MDS (PNAS 2009; 106: 12974). We now show that shRNA suppression of these genes to levels commensurate with haplodeficiency reinforces p53 accumulation, and that treatment with LEN promotes MDM2-mediated p53 degradation to transition del(5q) clones to G2/M arrest. We hypothesized that emergence of resistance to LEN in del(5q) MDS arises from two possible mechanisms: (1) up-regulation of haplodeficient drug targets or compensatory isotypes, or (2) inactivating mutations of the TP53 or CDC25C genes. Methods:To investigate mechanisms of LEN resistance, we studied sequential bone marrow (BM) specimens obtained at baseline (BL), response to treatment (TR) and treatment failure (TF) from 12 LEN treated patients with Low/INT-1 risk, transfusion-dependent del(5q) MDS. Eleven patients achieved clonal suppression and transfusion independence; 7 patients developed clinical drug resistance with primary clonal recovery. Immunohistochemical (IHC) staining for cdc25-C, -A and -B; PP2A–Ca and p53 were performed using a biotin-streptavidin-horseradish peroxidase method and compared to 6 age-matched controls; intensity of cytoplasmic or nuclear staining in hematopoietic elements was recorded after blinded review. DNA and RNA were extracted from cryopreserved BM mononuclear cells (BM-MNC) or fixed paraffin blocks from BM clot and biopsy sections. Expression of CDC25C splice variants was assessed by RT-PCR and total gene expression by real time (QT)-PCR. Exonic DNA encoding the catalytic [exons 8–14] and nuclear export domains [exon 11] of CDC25C and the DNA-binding domain of TP53 [exons 4–9] was sequenced for gene mutation analysis. Differences in mean values were compared by paired t-test. Results:P53 immunostaining was significantly higher in del(5q) BL specimens compared to controls ( relative expression [RE] 9.6 vs. 0.25; P =0.007). An admixture of nuclear and cytoplasmic staining for p53 and each cdc25 isotype was observed at BL that was largely restricted to erythroid precursors, whereas at TR cdc25-C and -A expression was primarily cytoplasmic, consistent with drug-induced nuclear exclusion. At TR, RE of only cdc25C (BL, 75 vs. TR, 49; P=0.05) and PP2A (29.2 vs. 12.3; P=0.025) was significantly reduced; whereas at TF cdc25C (TR, 43 vs. TF, 166; P=0.003), cdc25A (42.4 vs. 150; P=0.006), PP2A (7.3 vs. 65.6; P=0.028) and p53 (0.92 vs. 25.4; P=0.024) RE significantly increased. Nuclear localization of cdc25C and p53 but not cdc25A predominated at TF, consistent with escape from cdc25C inhibition. QT-PCR confirmed transcriptional up-regulation of CDC25C at TF with a mean 8.8-fold increase in gene expression vs. BL. DNA sequencing revealed no acquisition of somatic mutations within the CDC25C and TP53 exons studied [n=5]. Conclusions:Secondary resistance to LEN in del(5q) MDS is associated with over-expression and activation of the haplodeficient drug-inhibitable phosphatases, cdc25C and PP2A, with consequent restoration of wt-p53 activation. Absence of gene mutations within the coding exons analyzed suggests that transcriptional compensation alone is responsible for drug resistance. Novel agents targeting transcriptional repression of CDC25C may restore LEN sensitivity and merit investigation in drug resistant del(5q) MDS. Disclosures:List:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Komrokji:Celgene: Research Funding, Speakers Bureau. Lancet:Celgene: Research Funding. Maciejewski:Esai: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Mdm2/Mdmx and Mdm2/c‐Abl Interactions are Accompanied by Reductions in Mdm2/p53 interaction in Zinc‐Depleted Human Hepatoblastoma Cells

Mdmx has been shown to interact with Mdm2, interfere with Mdm2 degradation, and inhibit Mdm2‐mediated p53 degradation. The influence of zinc deficiency on the interaction of Mdm2/p53, Mdm2/Mdmx, and cAbl/Mdm2 was examined in human hepatoblastoma (HepG2) cells. Cells were cultured in zinc‐deficient (ZD0.2 and ZD0.4 with 0.2 and 0.4 uM zinc, respectively) or zinc normal (ZN with 4 uM zinc) medium. Mdm2/p53, Mdmx/Mdm2, and c‐Abl/mdm2 interactions were detected by Immunoprecipitation assays. Interestingly, Mdm2/p53 interaction was significantly decreased in ZD0.2 and ZD0.4 cells as compared with ZN cells. In addition, Mdm2/p53 interaction was significantly lower in ZD0.2 cells as compared with ZD0.4 cells. Importantly, the amount of Mdmx bound to Mdm2 was increased in ZD0.2 and ZD0.4 cells as compared with ZN cells. c‐Abl/Mdm2 interaction was higher in ZD0.2 and ZD0.4 cell than in ZN cells. Moreover, p‐Tyr of Mdm2, which is known to impair the interaction of Mdm2 with p53, was found to be higher in ZD cells as compared with ZN cells. Zinc depletion in ZD0.2 and ZD0.4 cells increased the phosphorylation of p53 at serine 15 and 392 but not at serine 20. In conclusion, we believe that this increase in phosphorylation of p53 at serine 15 is the most direct way that zinc deficiency stabilizes p53 and enhances the accumulation of nuclear p53 and Mdm2 in zinc deficient HepG2 cells. Moreover, Mdm2 interacts with Mdmx or c‐Abl and may also contribute to the accumulation of nuclear p53 and Mdm2 in zinc deficient cells.