Activation Of P53 Target Genes Research Articles

Expression levels of hnRNPK and p21WAF1/CIP1 are associated with resistance to radiochemotherapy independent ofp53 pathway activation in rectal adenocarcinoma.

Ionizing radiation (IR) is frequently applied in the treatment of rectal adenocarcinoma, however, there is marked variance in the response to radiochemotherapy between individual tumors. In our previous investigations, it was shown that the overexpression of heterogeneous nuclear ribonucleoprotein K (hnRNP K) confers radioresistance to malignant melanoma and colorectal carcinoma (CRC) in vitro, however, the underlying mechanism remains to be elucidated. As hnRNP K, a p53 binding partner and cofactor for the transcriptional activation of p53 target genes, is overexpressed in CRC, the present study investigated the possible radioprotective effect of the hnRNP K/p53-induced upregulation of p21 (also known as WAF1/CIP1) in rectal adenocarcinoma. Immunohistochemistry was performed for hnRNP K, p53 and p21 in a series of 68 consecutive cases of rectal adenocarcinoma with full molecular characterization following radiochemo-therapy and 14 corresponding pre-therapeutic biopsies, and the results were correlated with clinicopathological characteristics and the percentage of vital tumor cells following therapy. In addition, pathway analyses, protein immunoprecipitation, western immunoblotting and immunofluorescence microscopy were performed to identify dysregulated kinase signaling and hnRNP K targets upon exposure of CRC cells to IR. Although the fraction of vital tumor cells upon neoadjuvant therapy was significantly higher in hnRNP K/p21-positive tumors (P=0.0047 and P=0.0223, Students’ t-test), no significant association was found between the protein expression levels of hnRNP K, p53 and p21 (P>0.05, χ2 test). Irradiation enhanced apoptotic pathway activation via p53/CHK2 phosphorylation and poly (ADP-ribose) polymerase cleavage, and induced the overexpression and interaction of hnRNP K and p53. However, p53 Ser15-phosphorylation was independent of the presence of hnRNP K, and there was no measurable effect of hnRNP K on the expression of p21 in vitro. Taken together, the results of the present study support a radioprotective role for hnRNP K, which may be mediated through an interaction with p53, however, this effect appears to be independent of the hnRNP K/p53-induced upregulation of p21 in rectal adenocarcinoma.

Copper and ROS mediation in glycoxidation of a human serum albumin (HSA) model peptide: Advanced Glycation End‐Products (AGEs) generation and damage in motor neuron cells

Glucose, in the presence of reactive oxygen species (ROS), acts as an as an oxidative agent and drives deleterious processes in Diabetes Mellitus. We have studied the mechanism and the toxicological effects of glucose‐dependent glycoxidation reactions driven by copper and ROS, using a model peptide based on the exposed sequence of Human Serum Albumin (HSA) and containing a lysine residue susceptible to copper complexation. The main products of these reactions are Advanced Glycation End‐products (AGEs).In vitro studies of glycation and glycoxidation often use Human Serum Albumin (HSA) as a model protein. HSA has several metal‐binding sites and it is a protein useful for studies of glycation/ glycoxidation, since it has several lysine and arginine residues, which have free amino groups in their side chains. We have studied the effects of copper ions and hydroxyl radicals, on the glycoxidation of a mimetic HAS peptide that includes the lysine residue present in the three homologous domain structures repeated in the molecule (I, II and III) and in the A and B subdomains. The model peptide is based on an exposed sequence in HSA that contains a lysine residue susceptible to complexation with copper ions .Pyrraline condensed on the model peptide, generating a Modified Peptide (MP). These products were isolated, purified, and tested on cultured motor neuron cells. We observed DNA damage, enhancement of membrane roughness, and formation of domes. We evaluated nuclear abnormalities by the cytokinesis‐blocked micronucleus assay and we measured cytostatic and cytotoxic effects, chromosomal breakage, nuclear abnormalities, and cell death. AGEs formed by glycoxidation caused large micronucleus aberrations, apoptosis, and large‐scale nuclear abnormalities, even at low concentrations. We also observed increases in many proteins by Western blot, as NF‐κB, cyclin D1 and caspase 3 and 9 during cell treatments with MP. Activation of NF‐κB generally involves phosphorylation of IκB by the IKB kinase complex (IKK), resulting in IκB degradation. The regulation of NF‐κB genes affects apoptosis, cell adhesion, proliferation, immune responses, inflammation, and cellular stress response. NF‐κB activity can be regulated by oncogenes and tumor suppressors and may contribute to the pathology of inflammatory diseases and cancers. Activation of p53 can inhibit cyclin D1 expression by inducing association of the p52 NF‐κB subunit with co‐repressor complexes. In some cell lines, p52 is recruited directly to p53‐regulated promoters, leading to repression or activation of p53 target genes. Through this route, p52 may regularize cell cycle arrest dependent on p53 and apoptosis.We concluded that accelerated formation of AGEs through glycoxidation reactions could serve as a biomarker of neurodegeneration in oxidative conditions such as AD and possibly in carcinogenesis.Support or Funding InformationFAPESPCNPqCAPESThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Tumor Suppressor p53 in Mucosal Melanoma of the Head and Neck.

Despite worldwide prevention programs, the incidence for cutaneous melanoma is continuously increasing. Mucosal melanoma (MM) represents a rare but highly aggressive phenotype of common melanoma with predilection in the sinonasal system. Far away from ultraviolet sun exposure, the molecular mechanisms underlying tumorigenesis and the highly aggressive clinical behavior are poorly understood. In many solid malignomas of the head and neck region, p53 tumor suppressor functions as oncogene due to p53 protein stabilizing mutation. Interestingly, the vast majority of MM demonstrates constitutively expressed p53 protein, with protein stabilizing mutations being rare. Abrogated activation of p53 target genes results in derogation of the apoptotic signal cascade and contributes to the strong resistance against chemotherapeutic agents activating p53 dependent apoptosis. The current review illustrates the role of p53 and its pathway in MM.

P53 partners with RNA in the DNA damage response.

Stabilization of p53 protein is a key step in the cellular response to DNA damage. A new study describes a long noncoding RNA, DINO, transcribed from the CDKN1A promoter region that induces stabilization of p53 protein and promotes efficient activation of p53 target genes in response to DNA damage.

NF-Y loss triggers p53 stabilization and apoptosis in HPV18-positive cells by affecting E6 transcription

The expression of the high risk HPV18 E6 and E7 oncogenic proteins induces the transformation of epithelial cells, through the disruption of p53 and Rb function. The binding of cellular transcription factors to cis-regulatory elements in the viral Upstream Regulatory Region (URR) stimulates E6/E7 transcription. Here, we demonstrate that the CCAAT-transcription factor NF-Y binds to a non-canonical motif within the URR and activates viral gene expression. In addition, NF-Y indirectly up-regulates HPV18 transcription through the transactivation of multiple cellular transcription factors. NF-YA depletion inhibits the expression of E6 and E7 genes and re-establishes functional p53. The activation of p53 target genes in turn leads to apoptotic cell death. Finally, we show that NF-YA loss sensitizes HPV18-positive cells toward the DNA damaging agent Doxorubicin, via p53-mediated transcriptional response.

Colorectal cancer cells display chaperone dependency for the unconventional prefoldin URI1.

Chaperone dependency of cancer cells is an emerging trait that relates to the need of transformed cells to cope with the various stresses associated with the malignant state. URI1 (unconventional prefoldin RPB5 interactor 1) encodes a member of the prefoldin (PFD) family of molecular chaperones that acts as part of a heterohexameric PFD complex, the URI1 complex (URI1C), to promote assembly of multiprotein complexes involved in cell signaling and transcription processes. Here, we report that human colorectal cancer (CRCs) cell lines demonstrate differential dependency on URI1 and on the URI1 partner PFD STAP1 for survival, suggesting that this differential vulnerability of CRC cells is directly linked to URI1C chaperone function. Interestingly, in URI1-dependent CRC cells, URI1 deficiency is associated with non-genotoxic p53 activation and p53-dependent apoptosis. URI1-independent CRC cells do not exhibit such effects even in the context of wildtype p53. Lastly, in tumor xenografts, the conditional depletion of URI1 in URI1-dependent CRC cells was, after tumor establishment, associated with severe inhibition of subsequent tumor growth and activation of p53 target genes. Thus, a subset of CRC cells has acquired a dependency on the URI1 chaperone system for survival, providing an example of ‘non-oncogene addiction’ and vulnerability for therapeutic targeting.

A Novel Conditional Knockout of the Diamond Blackfan Anemia Gene Rpl11 Shows Failure of Erythropoiesis, a Marked Increase in BFU-E Progenitors By Phenotype That Proliferate Poorly in Culture, and Activation of p53 Target Genes

Diamond Blackman Anemia (DBA) is a severe erythroid hypoplasia that usually presents during the first year of life. Most cases are due to heterozygous mutations in ribosomal protein genes including RPS19, RPL5 and RPL11. Ribosomal stress-induced p53 activation is thought to play a major role in DBA, leading to apoptosis and/or cell cycle arrest in the erythroid lineage. This response is dependent on accumulation of RPL5 and RPL11 subunits that bind and sequester MDM2, the ubiquitin ligase that naturally degrades p53. Thus, it is difficult to understand how this pathway can operate in the presence RPL5 or RPL11 mutations. Here we show that Rpl11 haploinsufficiency in mice leads to severe anemia characteristic of DBA and, surprisingly, p53 activation in the bone marrow.We created a conditional Rpl11 mouse with loxP sites that flank exon 2. These animals were crossed with MxCre heterozygotes, and treated with pIpC. After three weeks, Rpl11+/- animals became severely anemic, with increased MCV, decreased reticulocyte counts and normal leukocyte and platelet counts. Sacrifice of survivors after 4 months showed increased spleen weight due to the murine stress erythroid response. FACS analysis of bone marrow cells showed a marked increase in BFU-E and relative decrease in CFU-E in Rpl11+/- animals. However, methylcellulose colony forming assays revealed a decrease in large BFU-E colonies from Rpl11+/- animals, coupled with an increase in smaller, atypical BFU-E colonies, which fail to proliferate at a normal rate. To identify transcriptional signatures associated with severe anemia in Rpl11+/- animals, we sequenced cDNA libraries from total BM of induced Rpl11+/- mice and identified 508 differentially expressed genes relative to controls. Unexpectedly, p53 pathway genes were significantly upregulated in Rpl11+/- mice, which may suggest that Rpl5 accumulation alone or with haploinsufficient amounts of Rpl11 can sequester MDM2, or that p53 activation occurs through an alternative mechanism. In conclusion, we created a unique genetic heterozygous Rpl11 conditional knockout mouse that recapitulates the severe macrocytic anemia of DBA. We wish to use this model to identify novel molecular mechanisms of pathogenesis and test emerging therapies. DisclosuresEbert:Genoptix: Consultancy, Patents & Royalties; Celgene: Consultancy; H3 Biomedicine: Consultancy.

Combination of Dasatinib with Conventional Chemotherapy Is Associated with a High Response Rate in High Risk Acute Myeloid Leukemia (AML)

Background: High risk AML patients (pts) have poor outcome with low complete remission (CR) rate and long term survival despite intensive chemotherapy.Tyrosine kinases play an important role in AML pathogenesis. Pre-clinical studies performed in our center (Blood. 2013 122:1900) have shown that Src family tyrosine kinases (SFK) including Lyn, Hck and Fgr are abnormally activated in AML compared to normal hematopoietic stem cell and progenitor cells. Other studies have shown that the c-Kit receptor is over-activated in a subset of AML pts and contributes to abnormal leukemia cell growth. We further show that the small molecule SFK and c-Kit inhibitor dasatinib reduces proliferation and survival of AML stem and progenitor cells. Importantly, dasatinib enhances the sensitivity of AML stem and progenitor cells to chemotherapeutic agents by inhibiting Akt signaling, increasing mdm2 phosphorylation and enhancing p53 activity in AML cells. Based on this data we conducted a phase I/II clinical study of the combination of dasatinib with conventional cytarabine-idarubicin ("7+3")-based induction chemotherapy in high-risk AML.Methods: Between September 20013 and July 2015, 18 adult AML pts were enrolled in the study. Eligibility criteria were high-risk AML, age >18 years, and suitability for intensive therapy. High risk AML was defined by one of the following criteria: older age (> 60 yrs), poor-risk cytogenetic and molecular abnormalities (ELN criteria), secondary disease (AML evolving from myelodysplasia or myeloproliferative neoplasm), therapy related (t-AML), or relapsed/refractory.This Phase I study used a 3 + 3 dose escalation design for dasatinib while keeping a fixed dose of cytarabine and idarubicin (cytarabine 200 mg/m2 CIV days 1-7, idarubicin 12 mg/m2 IV days 1-3). Dasatinib was started at a dose of 70 mg (dose level 1; DL-1) days 1-7, and escalated to 100 mg orally days 1-7 (DL 1). Pts who failed to achieve CR received second re-induction with the same regimen. Pts who achieved CR received consolidation high dose cytarabine and/or allogeneic stem cell transplantation (SCT) based on donor availability.Results: Of the 18 pts enrolled on the study, 7 pts (39%) had secondary AML, 5 pts (28%) relapsed AML, 2 pts (11%) t-AML, and 4 (22%) were newly diagnosed older AML pts (one with complex karyotype, one with trisomy 8 and one cytogenetically normal with FTL3-ITD). The median age of all pts was 62 yrs (range 27-73). Of the 18 pts, 13 pts are currently evaluable for response; of which 10 (77%) achieved CR/Cri. Of the 5 non-evaluable pts, one pt withdrew from study after 2 days of therapy, one pt was taken off study after 3 days due to cytarabine neurotoxicity, one pt died of intracranial hemorrhage before the day 14 bone marrow biopsy, one pt had therapy interrupted secondary to pneumonia and sepsis, and one is still receiving therapy. None of the pts required more than one induction to achieve CR. Of the 10 pts who achieved CR/Cri, 8 patients went on to receive allogeneic SCT. As expected, the most commonly reported grade 3 and 4 adverse events (AE) were anemia 50%, thrombocytopenia 44%, neutropenia 38%, and fatigue 27%. The most common grade 1-2 AE were GI toxicities 61% and rash 33%.Correlative studies performed on blood samples obtained from pts on day 3 after initiation of treatment (n=9) demonstrated significant decrease in of SRC activity (as indicated by reduction in phospho-SRC levels on immunoblotting, 0.52±0.11 of control, p=0.01), and increased expression of p53-target genes as evaluated by Q-RT-PCT [including Puma (16.2±6.9 fold, p=0.015), P21 (4.9±1.1 fold, p=0.004), DR5 (3.4±0.9, p=0.004), Bax (3.7±0.9 fold, p=0.001) and HDM2 (2.1±0.5, p=0.02)].Conclusion: Combination of dasatinib with conventional "7+3" induction chemotherapy is feasible in high-risk AML and leads to higher CR rate compared to historical data without increase in toxicity rate, allowing more pts to receive allogeneic transplantation. Correlative laboratory studies are consistent with pre-clinical studies suggesting that this combination is associated with significant inhibition of SRC activity and enhanced activation of p53-target genes. DisclosuresDos Santos:Amgen: Employment. Stein:Amgen: Speakers Bureau. Chen:Seattle Genetics: Consultancy, Research Funding, Speakers Bureau; Millennium: Consultancy, Research Funding, Speakers Bureau; Merck: Consultancy, Research Funding; genentech: Consultancy, Speakers Bureau. Khaled:Sequenom: Research Funding.

Alpha-enolase is upregulated on the cell surface and responds to plasminogen activation in mice expressing a ∆133p53α mimic.

The p53 protein is a master regulator of the stress response. It acts as a tumor suppressor by inducing transcriptional activation of p53 target genes, with roles in apoptosis, cell cycle arrest and metabolism. The discovery of at least 12 isoforms of p53, some of which have tumor-promoting properties, has opened new avenues of research. Our previous work studied tumor phenotypes in four mouse models with different p53 backgrounds: wild-type p53, p53 null, mutant p53 lacking the proline domain (mΔpro), and a mimic for the human Δ133p53α p53 isoform (Δ122p53). To identify the major proteins affected by p53 function early in the response to DNA damage, the current study investigated the entire proteome of bone marrow, thymus, and lung in the four p53 models. Protein extracts from untreated controls and those treated with amsacrine were analyzed using two-dimensional fluorescence difference gel electrophoresis. In the bone marrow, reactive proteins were universally decreased by wild-type p53, including α-enolase. Further analysis of α-enolase in the p53 models revealed that it was instead increased in Δ122p53 hematopoietic and tumor cell cytosol and on the cell surface. Alpha-enolase on the surface of Δ122p53 cells acted as a plasminogen receptor, with tumor necrosis factor alpha induced upon plasminogen stimulation. Taken together, these data identified new proteins associated with p53 function. One of these proteins, α-enolase, is regulated differently by wild-type p53 and Δ122p53 cells, with reduced abundance as part of a wild-type p53 response and increased abundance with Δ122p53 function. Increased cell surface α-enolase on Δ122p53 cells provides a possible explanation for the model’s pro-inflammatory features and suggests that p53 isoforms may direct an inflammatory response by increasing the amount of α-enolase on the cell surface.

The selective activation of p53 target genes regulated by SMYD2 in BIX-01294 induced autophagy-related cell death.

Transcription regulation emerged to be one of the key mechanisms in regulating autophagy. Inhibitors of H3K9 methylation activates the expression of LC3B, as well as other autophagy-related genes, and promotes autophagy process. However, the detailed mechanisms of autophagy regulated by nuclear factors remain elusive. In this study, we performed a drug screen of SMYD2-/- cells and discovered that SMYD2 deficiency enhanced the cell death induced by BIX01294, an inhibitor of histone H3K9 methylation. BIX-01294 induces accumulation of LC3 II and autophagy-related cell death, but not caspase-dependent apoptosis. We profiled the global gene expression pattern after treatment with BIX-01294, in comparison with rapamycin. BIX-01294 selectively activates the downstream genes of p53 signaling, such as p21 and DOR, but not PUMA, a typical p53 target gene inducing apoptosis. BIX-01294 also induces other autophagy-related genes, such as ATG4A and ATG9A. SMYD2 is a methyltransferase for p53 and regulates its transcription activity. Its deficiency enhances the BIX-01294-induced autophagy-related cell death through transcriptionally promoting the expression of p53 target genes. Taken together, our data suggest BIX-01294 induces autophagy-related cell death and selectively activates p53 target genes, which is repressed by SMYD2 methyltransferase.

Inhibition of HDAC8 Reactivates p53 and Abrogates Leukemia Stem Cell Activity in CBFβ-SMMHC Associated Acute Myeloid Leukemia

The inv(16)-created CBFβ-SMMHC fusion protein inhibits differentiation of hematopoietic stem and progenitor cells (HSPCs) and creates pre-leukemic populations predisposed to acute myeloid leukemia (AML) transformation. However, the molecular mechanism underlying the leukemogenic function of CBFβ-SMMHC has been elusive. Given the low TP53 mutation rate in AML, alternative mechanisms disrupting p53 function are expected. We showed thatCBFβ-SMMHC impairs p53 acetylation and p53 target gene activation through formation of an aberrant protein complex with p53 and HDAC8 (Blood, 120: A772; 122(21): 224). We now show that CBFβ-SMMHC binds to p53 and HDAC8 independently through distinct regions and that HDAC8 mediates the deacetylation of p53 associated with CBFβ-SMMHC. In addition, we generated mice carrying a floxed Hdac8 (Hdac8f) allele and crossed with Cbfb56M/+/Mx1-Cre (Kuo YH et al, Cancer Cell 2006). Deletion of Hdac8 signifiacntly (p<0.0001) reduced the incidence of AML and prolonged disease-free survival. Pharmacologic inhibition of HDAC8 activity with HDAC8-selective inhibitors (HDAC8i) reactivates p53 and selectively induces apoptosis of inv(16)+ AML CD34+ cells while sparing normal HSPCs. To test the effect of HDAC8i on LSC engraftment and leukemia-initiating capacity, we generated Cbfb56M/+/Mx1-Cre mice with a Cre-reporter line expressing tdTomato fluorescence protein following Cre-mediated recombination. AML cells (dTomato+/cKit+) treated with HDAC8i (22d) ex vivo showed reduced engraftment (p=0.025) and enhanced survival (p=0.025) in transplanted mice. To examine whether HDAC8i 22d treatment affects the engraftment capacity on surviving cells, we transplanted equal number (2 x 106) of AML cells treated with either 22d or vehicle in another cohort of mice (n=4). We show that HDAC8i 22d treatment reduced the engraftment of dTomato+/cKit+ AML cells and enhanced survival, suggesting that the engraftment capacity is altered in addition to reducing AML cell survival. We next performed preclinical studies to determine the efficacy of in vivo administration of HDAC8i 22d. AML transplanted mice were randomized into two groups, one group treated with vehicle and the other treated with HDAC8i 22d for 2 weeks. Flow cytometry analysis revealed significantly reduced frequency (p=0.0097) and number (p=0.0101) of dTomato+/cKit+ AML cells in the bone marrow and spleen of 22d treated mice compared to vehicle treated group. To further assess the impact on LSC activity, we transplanted bone marrow cells from these treated mice into secondary recipients and analyzed for AML engraftment. Significant reduction in the frequency (p<0.0001) and the number (p=0.0006) of dTomato+/cKit+ AML cells was observed in the bone marrow and spleen. Furthermore, HDAC8i 22d treated transplants showed no signs of leukemia while vehicle treated transplants are moribund with aggressive AML. These results indicate that HDAC8 inhibition by 22d treatment effectively eliminates engraftment and leukemia-initiating capacity of AML LSCs.In conclusion, our studies identify a novel post-translational p53-inactivating mechanism and demonstrate selective HDAC8 inhibition as a promising approach to target inv(16)+ AML LSCs. DisclosuresNo relevant conflicts of interest to declare.

Abstract 468: Cancer cells with G/G mdm2 SNP309 have compromised transcriptional elongation of p53 target genes

Abstract MDM2 protein is often found over-expressed in cancers with wild-type p53. A single nucleotide polymorphism (T to G) near the mdm2 P2 promoter, mdm2 SNP309, leads to MDM2 overexpression. This polymorphism is associated with accelerated tumor formation, decreased sensitivity to DNA damage treatment and compromised p53 transcriptional activity. Two G/G SNP309 cell lines MANCA (Burkitt lymphoma) and A875 (melanoma) express wild-type p53 protein not readily degraded by MDM2. This wild-type p53 does not activate target genes even after DNA damage. During the blocked transcription, MDM2 and p53 co-localize at p53 response elements on chromatin. Therefore, we hypothesized that MDM2 localized with p53 on the chromatin was the contributing factor for the compromised p53 transcriptional activity. We explored the mechanism for compromised p53 transcriptional activity in these G/G SNP309 cancer cell lines. When compared to fully activated wild-type p53 in ML-1 cells treated with etoposide, MANCA and A875 cell lines had comparable recruitment of total and initiated RNA pol II at transcription start sites (TSS) for p21 and puma genes. This indicated that G/G SNP309 cells had functional transcription initiation at p53 target genes after DNA damage treatment. We next assessed epigenetic modifications associated with active transcriptional elongation. Using H3K36 trimethylation (H3K36me3) as a hallmark for active elongation, we observed that ML-1 cells had a substantial increase in H3K36me3 after etoposide treatment at p21 and puma TSS that was not seen in MANCA or A875 cell lines. This lack of H3K36me3 suggested an inhibition in early transcriptional elongation. To address the participation of MDM2 in the elongation block we created a stable MDM2 knockdown MANCA cell line. Interestingly, MDM2 knockdown drastically increased MDMX protein levels and moderately increased activation of p53 target genes implying MDMX played a redundant role for MDM2. We are currently investigating the roles of MDM2 and MDMX in blocking transcriptional elongation. Interestingly, we have found that treatment of MANCA cells with low dose Actinomycin D reactivates the p53 pathway and studies are in progress to determine if this is through activation of transcriptional elongation. This work was supported by The Breast Cancer Research Foundation and The National Science Foundation (grant MCB-0744316) to J.B. M.R. was supported by the MBRS-RISE Program to Hunter College 3R25-GM060665. Citation Format: Melissa Rosso, Alla Polotskaia, Jill Bargonetti. Cancer cells with G/G mdm2 SNP309 have compromised transcriptional elongation of p53 target genes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 468. doi:10.1158/1538-7445.AM2014-468

Abstract 5138: Histone deacetylase 2 and N-Myc reduce p53 protein phosphorylation at serine 46 by repressing gene transcription of tumor protein 53-induced nuclear protein 1

Abstract Myc oncoproteins and histone deacetylases (HDACs) exert oncogenic effects by modulating gene transcription. Paradoxically, N-Myc induces p53 gene expression. Tumor protein 53-induced nuclear protein 1 (TP53INP1) phosphorylates p53 protein at serine 46, leading to enhanced p53 activity, transcriptional activation of p53 target genes and programmed cell death. We aimed to identify the mechanism through which N-Myc overexpressing p53 wild-type neuroblastoma cells acquired resistance to apoptosis.Gene and protein expression was analysed by real-time RT-PCR and immunoblot. Cell survival/death was examined by flow cytometry study of Annexin V-staining. The prognostic value of TP53INP1 expression in tumor tissues was investigated in three independent cohorts of neuroblastoma patients. TP53INP1 was one of the genes most significantly repressed by HDAC2 and N-Myc according to Affymetrix microarray gene expression datasets. HDAC2 and N-Myc reduced TP53INP1 gene expression by direct binding to the TP53INP1 gene promoter, leading to transcriptional repression of TP53INP1, p53 protein de-phosphorylation at serine 46, neuroblastoma cell proliferation and survival. Moreover, low levels of TP53INP1 expression in human neuroblastoma tissues correlated with high levels of N-Myc expression and poor patient outcome, and the BET bromodomain inhibitors JQ1 and I-BET151 reduced N-Myc expression and reactivated TP53INP1 expression in neuroblastoma cells. These findings identify TP53INP1 repression as an important co-factor for N-Myc oncogenesis, and provide further evidence for the potential application of BET bromodomain inhibitors in the therapy of N-Myc-induced neuroblastoma. Citation Format: Jeyran Shahbazi, Christopher J. Scarlett, Murray Norris, Bing Liu, Michelle Haber, Andrew E. Tee, Alice Carrier, Andrew V. Biankin, Wendy B. London, Glenn M. Marshall, Richard Lock, Tao Liu. Histone deacetylase 2 and N-Myc reduce p53 protein phosphorylation at serine 46 by repressing gene transcription of tumor protein 53-induced nuclear protein 1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5138. doi:10.1158/1538-7445.AM2014-5138

Histone deacetylase 2 and N-Myc reduce p53 protein phosphorylation at serine 46 by repressing gene transcription of tumor protein 53-induced nuclear protein 1.

Myc oncoproteins and histone deacetylases (HDACs) exert oncogenic effects by modulating gene transcription. Paradoxically, N-Myc induces p53 gene expression. Tumor protein 53-induced nuclear protein 1 (TP53INP1) phosphorylates p53 protein at serine 46, leading to enhanced p53 activity, transcriptional activation of p53 target genes and programmed cell death. Here we aimed to identify the mechanism through which N-Myc overexpressing p53 wild-type neuroblastoma cells acquired resistance to apoptosis. TP53INP1 was found to be one of the genes most significantly repressed by HDAC2 and N-Myc according to Affymetrix microarray gene expression datasets. HDAC2 and N-Myc reduced TP53INP1 gene expression by direct binding to the TP53INP1 gene promoter, leading to transcriptional repression of TP53INP1, p53 protein de-phosphorylation at serine 46, neuroblastoma cell proliferation and survival. Moreover, low levels of TP53INP1 expression in human neuroblastoma tissues correlated with high levels of N-Myc expression and poor patient outcome, and the BET bromodomain inhibitors JQ1 and I-BET151 reduced N-Myc expression and reactivated TP53INP1 expression in neuroblastoma cells. These findings identify TP53INP1 repression as an important co-factor for N-Myc oncogenesis, and provide further evidence for the potential application of BET bromodomain inhibitors in the therapy of N-Myc-induced neuroblastoma.

Proline, glutamic acid and leucine-rich protein-1 is essential for optimal p53-mediated DNA damage response

Proline-, glutamic acid- and leucine-rich protein-1 (PELP1) is a scaffolding oncogenic protein that functions as a coregulator for a number of nuclear receptors. p53 is an important transcription factor and tumor suppressor that has a critical role in DNA damage response (DDR) including cell cycle arrest, repair or apoptosis. In this study, we found an unexpected role for PELP1 in modulating p53-mediated DDR. PELP1 is phosphorylated at Serine1033 by various DDR kinases like ataxia-telangiectasia mutated, ataxia telangiectasia and Rad3-related or DNAPKc and this phosphorylation of PELP1 is important for p53 coactivation functions. PELP1-depleted p53 (wild-type) breast cancer cells were less sensitive to various genotoxic agents including etoposide, camptothecin or γ-radiation. PELP1 interacts with p53, functions as p53-coactivator and is required for optimal activation of p53 target genes under genomic stress. Overall, these studies established a new role of PELP1 in DDRs and these findings will have future implications in our understanding of PELP1's role in cancer progression.

Effects of TP53 mutational status on gene expression patterns across 10 human cancer types

Mutations in the TP53 tumour suppressor gene occur in half of all human cancers, indicating its critical importance in inhibiting cancer development. Despite extensive studies, the mechanisms by which mutant p53 enhances tumour progression remain only partially understood. Here, using data from the Cancer Genome Atlas (TCGA), genomic and transcriptomic analyses were performed on 2256 tumours from 10 human cancer types. We show that tumours with TP53 mutations have altered gene expression profiles compared to tumours retaining two wild-type TP53 alleles. Among 113 known p53-up-regulated target genes identified from cell culture assays, 10 were consistently up-regulated in at least eight of 10 cancer types that retain both copies of wild-type TP53. RPS27L, CDKN1A (p21(CIP1)) and ZMAT3 were significantly up-regulated in all 10 cancer types retaining wild-type TP53. Using this p53-based expression analysis as a discovery tool, we used cell-based assays to identify five novel p53 target genes from genes consistently up-regulated in wild-type p53 cancers. Global gene expression analyses revealed that cell cycle regulatory genes and transcription factors E2F1, MYBL2 and FOXM1 were disproportionately up-regulated in many TP53 mutant cancer types. Finally, > 93% of tumours with a TP53 mutation exhibited greatly reduced wild-type p53 messenger expression, due to loss of heterozygosity or copy neutral loss of heterozygosity, supporting the concept of p53 as a recessive tumour suppressor. The data indicate that tumours with wild-type TP53 retain some aspects of p53-mediated growth inhibitory signalling through activation of p53 target genes and suppression of cell cycle regulatory genes.

The p53 Reactivating Drug PRIMA-1Met Induces Apoptosis Of Myeloma Cells Via ROS Production

Patients with multiple myeloma (MM) carrying a del(17)(p13) (del17p) deletion and/or TP53 mutation at diagnosis have a shortened survival whatever the treatment received. By using a large and characterized collection of human myeloma cell lines (HMCLs), we have shown that TP53mutated/deleted cell lines are more resistant to melphalan and nutlin3a (MDM2 inhibitor) than TP53wild-type HMCLs (Surget S, Cancer Res 2012;72:4562). The absence of a functional p53 pathway prevents the induction of p53 target genes, including pro-apoptotic genes, and therefore impairs apoptosis. In cancer cells, p53 mutations are frequent and highly expressed mutant p53 proteins exert a dominant effect. Pharmacological drugs such as PRIMA-1Met/APR246, which has been recently evaluated in a phase I/II study (Lehmann S J Clin Oncol 2012;30:3633) offers an interesting opportunity to redirect the activity of mutant misfolded p53 proteins. With this aim, the efficiency of PRIMA-1Met was assessed in 22 HMCLs and 16 primary MM samples characterized for del17p. HMCLs were heterogeneously sensitive to PRIMA-1Met, with an LD50 median value of 35 µM (range 4->100 µM). We failed to observe any correlation with HMCL's TP53 status since the LD50 median values of TP53wt, TP53mut and TP53del HMCLs were not significantly different (p=0.1, Kruskal Wallis test) at respectively 21 µM (range 4-70, n=7), 45 µM (21->100, n=13) and 21 µM (8-33, n=2). Primary MM samples were also heterogeneously sensitive, with a median cell death value induced by 10 µM PRIMA-1Met of 51% (n=16, range 5-100%). The 5 del17p+ samples showed no significant difference in their sensitivity to PRIMA-1Met from the 11 del17p- (p=0.31 Mann Whitney). Moreover, the sensitivity of 2 TP53wt and 2 TP53mut HMCLs was unchanged upon p53 silencing, and PRIMA-1Met failed to increase the expression of such p53 target genes as p21, Puma or DR5 (TRAIL-R2) in both TP53wt and TP53mut/del HMCLs. By contrast, nutlin3a induced cell death and increased DR5 expression in all TP53wt but in none of the TP53mut/del HMCLs (p<0.001, n=22) and both cell death and DR5 expression increase was prevented upon p53 silencing. In primary samples, nutlin3a increased DR5 expression in del17p- (n=9, median fold-increase 1.29) but not in del17p+ samples (n=4, median fold-increase 0.90, p=0.05) while PRIMA-1Met increased DR5 expression in neither del17p- nor del17p+ samples (0.90 versus 0.96, p=0.94). Despite a lack of activation of p53 target genes, PRIMA-1Met induced apoptosis as shown by caspase 3 and PARP cleavages. Interestingly, PRIMA-1Met induced a burst of ROS (shown by DHE staining in flow cytometry), involved in cell death since the ROS scavengers NAC (5 mM), N-acetyl-L-cysteine, as well as 5 mM GSH-MEE (gluthatione reduced ethyl ester, data not shown), strongly inhibited cell death in both HMCLs and primary cells with respective median inhibitions of 98% (n=8, p=0.01) and 93% (n=11, p<0.001 Wilcoxon matched-pairs signed rank test). Reciprocally, 0.5 mM BSO (L-buthionine-sulfoximine), an irreversible inhibitor of gamma-glutamyl cysteine synthetase, which depletes GSH in situ and in turn increases ROS, strongly synergized with 10 µM PRIMA-1Met and overcame resistance to apoptosis in 91% of HMCLs (n=22) and in 100% of primary samples (n=6). The median value of HMCL cell death was respectively 92% for 10µM PRIMA-1Met plus 0.5 mM BSO versus 2% for 10 µM PRIMA-1Met (n=22, p<0.001) and 4% for 0.5 mM BSO (n=22, p<0.001), and that of primary samples was 88% for PRIMA-1Met /BSO versus 22% for PRIMA-1Met (n=6, p=0.03) or 1% for BSO (n=6, p=0.03, Wilcoxon matched-pairs signed rank test). Notably, cell death induced by nutlin3a was not inhibited by NAC nor did BSO synergize with nutlin3a, confirming that nutlin3a and PRIMA-1Met have distinct death inducing pathways. Interestingly, the sensitivity of HMCLs to PRIMA-1Met alone correlated to the expression of an enzyme involved in oxidative stress (p=0.04, Spearman test), the participation of which is currently under investigation. Finally, the 10 µM PRIMA-1Met plus 0.5 mM BSO combination was found to be effective in only 2 of 6 mantle cell lymphoma cell lines, suggesting that it could be cell-type specific. These data show that PRIMA-1Met in combination with BSO has a strong pro-apoptotic activity in MM, independently of the p53 pathway, and thus could be of interest for del17p+ patients refractory to current therapies. Disclosures:No relevant conflicts of interest to declare.

Selective Targeting Of Inv(16)+ AML Stem Progenitor Cells By Inhibiting HDAC8

Chromosomal inversion inv(16)(p13.1q22) which leads to the fusion of the transcription factor gene CBFb and the MYH11 gene, occurs in over 8% of acute myeloid leukemia (AML) cases. The fusion product CBFβ-SMMHC (CM) inhibits differentiation of hematopoietic stem and progenitor cells (HSPCs) and creates pre-leukemic populations predisposed to acute myeloid leukemia (AML) transformation. The mutations of tumor suppressor p53 occur in approximately half of all cases of human cancer, but TP53 mutations are relatively rare in inv(16) AML. We have previously shown that CM expression leads to reduced acetylation of p53 and impaired p53 target gene activation through formation of aberrant protein complex with p53 and HDAC8 (Blood, 2012,120: A772.). Here, we showed that CM interacts with p53 both in CM transformed mouse primary bone marrow cells as well as in AML stem and progenitor cells from inv(16) patients. When HDAC8 selective pharmacological inhibitor 22d directed against its catalytic sites (ChemMedChem 2012, 7:10, 1815-24;) were used to treat inv(16) mouse primary bone marrow progenitor cells and inv(16)+ CD34+ stem progenitor cells from patients, Ac-p53 levels were remarkably increased as shown by western blot. We further assessed the p53 target genes expression after HDAC8 inhibitor 22d treatment by qRT-PCR assay in inv(16)+ CD34+ stem progenitor cells (n=8), and observed variable levels of activation in p53 targets (Fold activation: p21:2.25-fold, hdm2:1.17-fold, 14-3-3σ: 3.12-fold, puma: 2.39-fold), indicating p53 was re-activated. Similar results were also shown in CM transformed mouse bone marrow progenitor cells. Importantly, we found that 22d treatment significantly inhibit the growth of inv(16)+ AML CD34+ cells (n=9) rather than normal CD34+ cells (n=7) , (AML IC50= 6.509 μM, vs Normal cells IC50=13.83 μM, p=0.0003). Meanwhile, 22d selectively induces apoptosis of inv(16)+ AML stem and progenitor cells while sparing normal HSPCs (AML LD50= 10.24 μM, vs NL LD50= 46.36 μM, p=0.001). To evaluate whether the effect of HDAC8i is mediated by p53, we knocked down p53 with a lentiviral vector expressing shRNA against p53 (or non-silencing shRNA) in AML CD34+ cells, and treated the cells with HDAC8 inhibitor 22d (5-20 µM). We showed that despite the inter-sample variability, knocking down p53 expression in all AML samples tested (n=3) led to reduced HDAC8i-induced apoptosis, suggesting that p53 contributes to the apoptosis effect induced by HDAC8i (22d) in inv(16)+ AML cells. Importantly, by taking advantage of our conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre), which develops AML under induced expression of CBFß-SMMHC (Cancer Cell, 2006, 9:1, 57-68), we were able to perform the ex vivo treatment assay by treating primary leukemic cells (marked with dTomato) with either DMSO (as vehicle control) or with HDAC8 inhibitor 22d (10μM) for 48h, followed by transplantion into congenic mice (control group n=8, treatment group n=7). We observed reduced short-term engraftment of leukemic cells that are treated with 22d (10 μM) at 4 weeks post-transplantation in the peripheral blood (Donor cell%: control group=5.99%, treatment group=0.178%, P=0.0093). Interestingly, engraftment of cord blood CD34+ cells at 16 weeks post-bone marrow transplantation was not reduced after treatment with 22d (10 μM) (human CD45+ %: control=66.2% versus treatment=63.4%, p=0.9), indicating the effect by HDAC8 inhibition is selective for leukemic cells. In conclusion, we have identified a novel mechanism whereby CBFβ-SMMHC inhibits p53 fucntion, and may further implicate inhibition of HDAC8 as a promising approach to selectively target inv(16)+ AML stem and progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Abstract 4300: The EEF1A2-PI3K-AKT-mTOR axis supports the protumorigenic function of MDM4 in human hepatocellular carcinoma.

Abstract Background &amp; Aims: The Mouse Double Minute homolog 4 (MDM4) is one of the main negative p53 regulators in mammalian cells and mutational inactivation of p53 is a rare event in Western hepatocellular carcinomas (HCCs). 1q gains promote the upregulation of MDM4 in HCC, leading to a negative regulation of p53 activity. However, additional mechanisms might be involved that explain the increased MDM4 activity observed in HCCs with balanced MDM4 gene locus. Here we aimed at the identification of post-transcriptional mechanisms involved in the upregulation of MDM4 in HCC. Methods: To investigate a potential role of PI3K-AKT-mTOR signaling on MDM4 activity, HCC cell lines were treated with small molecule inhibitors and siRNAs and expression changes as well as functional analyses were recorded. Expression changes of central pathway components were investigated in human liver samples (normal liver, peritumorous liver tissue, and HCC). An AKT transgenic mouse model was used to show the involvement of AKT pathway in the regulation of MDM4 in vivo. Results: The inhibition of both PI3K-AKT and mTOR signaling pathways resulted in reduced MDM4 protein levels in HCC cell lines, which was associated with the transcriptional activation of p53-target genes. Biochemical assays revealed that both AKT-mediated phosphorylation and ubiquitin-specific protease 2a (USP2a)-mediated deubiquitination protected MDM4 from proteasomal in human HCC cell lines. In addition, AKT transgenic mice showed increased MDM4 protein levels indicating that AKT signaling is involved in the stabilization of MDM4 protein in vivo. Furthermore, the Eukaryotic translational Elongation Factor 1 alpha 2 (EEF1A2), which is frequently upregulated in human HCC, sustained the PI3K-AKT-mTOR cascade both in vitro and in vivo. In human HCCs, a strong positive correlation between the overexpression of EEF1A2, pAKT, USP2a, and MDM4 was observed, which was associated with shorter survival of HCC patients. Conclusions: Our data demonstrate that the EEF1A2/PI3K/AKT/mTOR cascade inactivates wild-type p53 in human HCC through the stabilization of the MDM4 protooncogene via a post-transcriptional mechanism involving an AKT-mediated phosphorylation of MDM4 and USP2a-mediated de-ubiquitination. Since the sustained activation of the EEF1A2/PI3K/AKT/mTOR/MDM4 axis has impact on the survival probability of HCC patients, it may thus represent a promising therapeutic target. Citation Format: Rossella Pellegrino, Diego F. Calvisi, Olaf Neumann, Xin Chen, Chunmei Wang, Bernhard Radlwimmer, Sara Ladu, Frank Dombrowski, Matthias Evert, Peter Schirmacher, Thomas Longerich. The EEF1A2-PI3K-AKT-mTOR axis supports the protumorigenic function of MDM4 in human hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4300. doi:10.1158/1538-7445.AM2013-4300

TFIIB dephosphorylation links transcription inhibition with the p53-dependent DNA damage response

The general transcription factor II B (TFIIB) plays a central role in both the assembly of the transcription complex at gene promoters and also in the events that lead to transcription initiation. TFIIB is phosphorylated at serine-65 at the promoters of several endogenous genes, and this modification is required to drive the formation of gene promoter-3' processing site contacts through the cleavage stimulation factor 3' (CstF 3')-processing complex. Here we demonstrate that TFIIB phosphorylation is dispensable for the transcription of genes activated by the p53 tumor suppressor. We find that the kinase activity of TFIIH is critical for the phosphorylation of TFIIB serine-65, but it is also dispensable for the transcriptional activation of p53-target genes. Moreover, we demonstrate that p53 directly interacts with CstF independent of TFIIB phosphorylation, providing an alternative route to the recruitment of 3'-processing complexes to the gene promoter. Finally, we show that DNA damage leads to a reduction in the level of phospho-ser65 TFIIB that leaves the p53 transcriptional response intact, but attenuates transcription at other genes. Our data reveal a mode of phospho-TFIIB-independent transcriptional regulation that prioritizes the transcription of p53-target genes during cellular stress.