bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 3683: The stress response mediator ATF3 stabilizes p53 by competing with it for MDM2-mediated ubiquitination
Authors: Li Xingyao , Yan Chunhong 
Published: July 2016

Abstract
While the tumor suppressor p53 is crucial for the maintenance of genetic integrity upon DNA damage and oncogenic stresses, its level and activity are tightly controlled by a complex network largely mediated by the E3 ubiquitin ligase MDM2. MDM2 catalyzes the addition of ubiqutin chains to the C-terminus of p53 and is the major ubiquitin ligase to promote proteosomal degradation of p53. We previously demonstrated that ATF3, a common stress response mediator, can bind p53 and prevent it from MDM2-mediated ubiquitination. As a consequence, ATF3 can stabilize p53 upon DNA damage and prevent cells from oncogene-induced transformation by promoting p53-mediated senescence, in line with accumulating evidence supporting that ATF3 can act as a tumor suppressor. Interestingly, ATF3 is also a bona fide substrate of MDM2, and thus, the ATF3-MDM2 interaction can fine tune p53 activity in the DNA damage response. However, the mechanism by which ATF3 blocks p53 ubiquitination to activate the latter protein remains largely undefined. Efficient ubiquitination of p53 requires the binding of MDM2 to the N-terminus of p53, and the MDM2 binding to MDMX. Here we show that neither the p53-MDM2 interaction nor the MDM2-MDMX interaction is disrupted by the binding of ATF3 to p53 or MDM2. As both ATF3 and p53 are substrates of MDM2, we explored a possibility that ATF3 competes with p53 for MDM2-mediated ubiquitination. By in-vitro ubiquitination assays, we identified that the 5 lysine residues clustering in aa 100-115 of ATF3 are required for MDM2-mediated ubiquitination. Site-specific mutagenesis indicates that MDM2 mainly ubiquitinates ATF3 at lysine 107 (K107) and lysine 108 (K108) as MDM2 fails to add ubiquitin chains to ATF3 when these two residues were mutated to arginine. Importantly, while mutations at these two residues do not affect the ATF3 binding to MDM2 or p53, the ATF3 mutant devoid of ubiquitination fails to stabilize p53 and prevent p53 from MDM2-mediated ubiquitination. These results thus demonstrate that ATF3 can activate p53 by competing with the tumor suppressor for MDM2-mediated ubiquitination.
Citation Format: Xingyao Li, Chunhong Yan. The stress response mediator ATF3 stabilizes p53 by competing with it for MDM2-mediated ubiquitination. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3683.
Abstract 3687: p53 downregulates CRKL oncogene through miR-200
Authors: Tokino Takashi , Tamura Miyuki , Idogawa Masashi , Sasaki Yasushi 
Published: July 2016

Abstract
Tumor suppressive miRNAs targeting oncogenes are frequently downregulated in cancers, which leads to the activation of the oncogene pathways. Thus, tumor suppressive miRNAs as well as their target oncogenes are proposed to be useful for cancer treatment. The downregulation of the miR-200 family has been involved in the progression and metastasis in cancers. The miR-200 family consists of two gene clusters: miR-200b/200a/429 and miR-200c/141 are located on human chromosome 1 and 12, respectively. Here, we identified that p53 response elements are located around both clusters of miR-200s and confirmed miR-200s as transcriptional targets of p53. in silico analysis of miRNA target predicted an oncogene CRKL as a potential target for miR-200b/200c/429. Moreover, miR-200b/200c/429 inhibit the expression of CRKL mRNA and protein by directly targeting its 3’-UTR region. Importantly, endogenous CRKL expression was decreased in cancer cells with introduction of wild-type p53. Moreover, downregulation of CRKL by siRNA in cancer cells inhibits cell growth. Oncomine database shows that CRKL levels are overexpressed in a subset of cancer types. Furthermore, CRKL is significantly overexpressed in primary breast cancer tissues harboring mutant TP53. Our results demonstrate that p53-target miRNAs, miR-200b/200c/429 are negative regulators of the CRKL oncogene.
Citation Format: Takashi Tokino, Miyuki Tamura, Masashi Idogawa, Yasushi Sasaki. p53 downregulates CRKL oncogene through miR-200. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3687.
Abstract 3688: The C-terminal domain of tumor-derived mutant p53 is required for its oncogenic gain-of-function activity
Authors: Lee Caleb C. , Manfredi James 
Published: July 2016

Abstract
p53 is the most commonly mutated gene in human cancers. Unlike other tumor suppressors in which loss of protein expression is common, the vast majority of changes in p53 arise as point mutations. Tumor associated mutation hotspots are localized to the DNA binding domain, either in residues involved in direct DNA contact or those that regulate its conformation. In either case, such mutations have been shown to confer gain of function activity. This was first shown in soft agar assays in which over-expression of mutant p53 increased transformation of cells on a p53 null background. Furthermore, knock-in mouse models of mutant p53 showed alterations in tumor spectrum with the development of carcinomas not seen in p53 null mice. However, the mechanism by which mutant p53 actively aids in tumorigenesis remains unclear.
Although mutant p53 causes loss of sequence specific DNA binding via the core domain, it retains the ability to interact with multiple proteins including transcriptional cofactors shown to have tumor suppressor activity such as p300 and CBP. These interactions occur through multiple protein binding domains including the C-terminal domain of p53. Our laboratory and others have shown that C-terminal truncation of wildtype p53 results in multiple deficiencies. Binding of mutant p53 to p300/CBP via the C-terminus is therefore an attractive hypothesis to explain mutant p53 gain of function.
To determine the role of the C-terminus in mutant p53 gain of function activity, we have expressed constructs containing a C-terminal truncation in combination with varying hotspot mutations. Transfection of mutant p53 into a p53 null cell line was required for the formation of colonies in a soft agar assay, indicating an increase in tumorigenicity. Furthermore, C-terminal truncation of the mutant p53 abrogated this phenotype suggesting that this domain is required for mutant p53 gain of function. This was independent of expression levels as shown by immunoblot. We have also extended the findings of mutant p53 interaction with p300 by IP analysis of a panel of cell lines with varying p53 hotspot mutations. To confirm the role of the C-terminus in mutant p53 gain of function, we are developing a xenograft model to detect changes in tumorigenicity in vivo. These results will therefore explore the mechanism by which mutant p53 acts as an oncogene.
Citation Format: Caleb C. Lee, James Manfredi. The C-terminal domain of tumor-derived mutant p53 is required for its oncogenic gain-of-function activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3688.
Abstract 3699: Aspirin acetylates wild type and mutant p53 in colon cancer cells: Identification of aspirin acetylated sites on recombinant p53
Authors: Ai Guoqiang , Dachineni Rakesh , Kumar Ramesh D. , Marimuthu Srinivasan , Alfonso Lloyd F. , Bhat Jayarama G. 
Published: July 2016

Abstract
Aspirin's ability to inhibit cell proliferation and induce apoptosis in cancer cell lines is considered to be an important mechanism for its anticancer effects. We previously demonstrated that aspirin acetylated the tumor suppressor protein p53 at lysine 382 in MDA-MB-231 human breast cancer cells. Here, we extended these observations to human colon cancer cells, HCT 116 harboring wild type p53, and HT-29 containing mutant p53. Aspirin induced acetylation of p53 in both cell lines in a concentration-dependent manner. Aspirin-acetylated p53 was localized to the nucleus. In both cell lines, aspirin induced p21CIP1. Aspirin also acetylated recombinant p53 (rp53) in vitro suggesting that it occurs through a non-enzymatic chemical reaction. Mass spectrometry analysis and immunoblotting identified 10 acetylated lysines on rp53, and molecular modeling showed that all lysines targeted by aspirin are surface exposed. Five of these lysines are localized to the DNA-binding domain, four to the nuclear localization signal domain, and one to the C-terminal regulatory domain. Our results suggest that aspirin's anticancer effect may involve acetylation and activation of wild type and mutant p53 and induction of target gene expression. This is the first report attempting to characterize p53 acetylation sites targeted by aspirin.
Citation Format: Guoqiang Ai, Rakesh Dachineni, Ramesh D. Kumar, Srinivasan Marimuthu, Lloyd F. Alfonso, Jayarama G. Bhat. Aspirin acetylates wild type and mutant p53 in colon cancer cells: Identification of aspirin acetylated sites on recombinant p53. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3699.
Abstract 3706: p53 represses pyrimidine catabolic gene dihydropyrimidine dehydrogenase (DPYD) expression following thymidylate synthase (TS) inhibition
Authors: Gokare Prashanth Ravishankar , Finnberg Niklas , Dai Jenny , Murphy Maureen , El-Deiry Wafik 
Published: July 2016

Abstract
Nucleotide catabolism by cancer cells can influence malignant behavior and intrinsic resistance to therapy. The rate-limiting enzyme in the pyrimidine catabolic pathway, dihydropyrimidine dehydrogenase (DPYD) contributes to the pharmacokinetics of fluorouracil (5-FU). Using in silico/chromatin-immunoprecipitation (ChIP) analysis we identify a conserved p53 DNA-binding site (p53BS) downstream of the DPYD gene with increased p53 occupancy following 5-FU. Histone H3K9 acetylation marks at the DPYD promoter is diminished concomitantly with reduced expression of DPYD mRNA and protein in a p53-dependent manner. Notably we find that the P72 allele of p53 suppresses DPYD expression more than the R72 p53 allele following 5-FU treatment in mouse embryo fibroblasts. Mechanistic studies reveal inhibition of DPYD expression by p53 is augmented following thymidylate synthase (TS) inhibition by 5-FU, methotrexate (MTX), raltitrexed and siRNA in cancer cells in vitro as well as in mice in vivo. DPYD repression by p53 is dependent on DNA-PK and ATM-signaling since pharmacologic targeting of these kinases reverses the transcriptional repression of DPYD by p53. Mice lacking p53 in their livers have increased conversion of 5-FU to 5-FUH2 in plasma and elicit a diminished 5-FU therapeutic response in syngeneic colorectal tumor xenografts as compared to littermates with an intact p53 allele consistent with increased DPYD-activity. Our data suggest that p53 plays an important role in controlling pyrimidine catabolism through its ability to regulate DPYD, particularly following metabolic stress imposed by nucleotide imbalance. The findings have implications for the toxicity and efficacy of the cancer therapeutic 5-fluorouracil.
Citation Format: Prashanth Ravishankar Gokare, Niklas Finnberg, Jenny Dai, Maureen Murphy, Wafik El-Deiry. p53 represses pyrimidine catabolic gene dihydropyrimidine dehydrogenase (DPYD) expression following thymidylate synthase (TS) inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3706.
Abstract 3720: Tissue-specific blockage of DNA damage responses sensitizes breast cancer chemotherapy
Authors: Xu Rong , Shen Haifa 
Published: July 2016

Abstract
Systemic chemotherapy is not effective at eradicating metastatic breast cancer, and approaches to improve the outcomes for these patients are urgently needed. The ideal method to enhance chemotherapy would involve targeting a protein that is differentially or exclusively expressed in cancer compared to normal tissue; however, such targets are rare. We sought to target the actively dividing breast cancer cells in the distant metastatic organs with tumor tissue-specific delivery of therapeutic siRNA that suppresses the expression of key DNA damage response (DDR) genes such as CHK1 and RAD51 in order to sensitize chemotherapy. Knockdown of CHK1 and RAD51 expression by the gene-specific siRNA oligos was confirmed in vitro in MDA-MB-231 human breast cancer cells. Treatment of nude mice bearing lung metastatic MDA-MB-231 tumors with tumor-enriched nanoparticle-packaged CHK1 or RAD5 siRNA did not significantly suppress tumor growth. Single agent treatment with biweekly 6 mg/kg doxorubicin treatment did not slow down growth of the metastatic tumors either. In contrast, combination treatment with 6 mg/kg doxorubicin and 15 μg CHK1 or RAD51 siRNA in nanoparticles dramatically inhibited tumor growth and extended animal survival. Our result supports the development of tumor tissue-enriched siRNA therapeutics targeting the DDR pathways to sensitize breast cancer therapy.
Citation Format: Rong Xu, Haifa Shen. Tissue-specific blockage of DNA damage responses sensitizes breast cancer chemotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3720.
Abstract 3726: Predicting cellular response to small molecule Mcl-1 antagonists
Authors: Hogdal Leah , Sensintaffar John , Arnold Allison , Goodwin Craig , Bian Zhiguo , Shaw Subrata , Tarr Chris , Lee Taekyu , Olejniczak Edward , Fesik Stephen 
Published: July 2016

Abstract
Myeloid cell leukemia -1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family which many cancers overexpress and depend on for survival. Mcl-1 inhibits apoptosis by binding to Bax and Bak, inhibiting the effectors of apoptosis from oligomerizing at the outer membrane of the mitochondria to promote cell death. Due to its role in cancer and resistance development to conventional chemotherapies, efforts to potently and specifically target MCL-1 have been ongoing. We have discovered novel small molecule inhibitors that selectively disrupt Mcl-1-Bim binding in the sub-nanomolar range. Anti-proliferative activities of our leading Mcl-1 inhibitors were assessed in a panel of hematological and solid cancer cell lines. We found that our compound promoted apoptosis in a subset of cell lines tested. As there was a variable response, we used BH3 profiling, which is a functional assay that rapidly measures dependence on any of the anti-apoptotic Bcl-2 family proteins for an individual sample to predict in vitro sensitivity to our Mcl-1 antagonists. Using the MS1 peptide, a known potent and selective Mcl-1 binder as a positive control in the BH3 profiling assay, we found a correlation between the BH3 profiling measurement of anti-apoptotic dependence to Mcl-1 and increased cytotoxicity caused by our compounds. These studies demonstrate that our compounds promote apoptosis through a selective inhibition of Mcl-1 mechanism of action and supports further investigation of our compounds in in vivo models of cancer.
Citation Format: Leah Hogdal, John Sensintaffar, Allison Arnold, Craig Goodwin, Zhiguo Bian, Subrata Shaw, Chris Tarr, Taekyu Lee, Edward Olejniczak, Stephen Fesik. Predicting cellular response to small molecule Mcl-1 antagonists. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3726.
Abstract 3727: Small molecule Mcl-1 inhibitors induce apoptosis and death in multiple cancer subtypes in vitro
Authors: Sensintaffar John L. , Arnold Allison , Goodwin Craig , Hogdal Leah , Shaw Subrata , Tarr James C. , Lee Taekyu , Olejniczak Edward , Fesik Stephen W. 
Published: July 2016

Abstract
Mcl-1 is a member of the Bcl-2 family of proteins that play a major role in conferring resistance to apoptosis in cancer cells. Inhibiting Mcl-1 using peptides or RNAi has been shown to induce apoptosis in a broad array of cancer cell lines in numerous studies, making Mcl-1 a compelling target for anticancer therapy. We have discovered potent and selective small molecule Mcl-1 inhibitors that bind to the BH3 binding site of Mcl-1 with sub nanomolar affinities. These agents rapidly induced apoptosis in the Mcl-1 dependent NCI-H929 myeloma cell line, as demonstrated by mitochondrial membrane depolarization, caspase activation, and decreased viability. We measured the anti-proliferative activity of our compounds in cell lines from several cancer subtypes and found a broad spectrum of sensitivity to Mcl-1 inhibition. In cell lines that were resistant to Mcl-1 antagonists, combination with the dual Bcl-2/Bcl-xL inhibitor ABT-263 (navitoclax) greatly enhanced the activity of both compounds. These findings demonstrate that pharmacologic inhibition of Mcl-1 as a single agent or in combination with other cancer therapeutic agents is an effective way to modulate the intrinsic apoptotic pathway and promote cell death in cancer cells.
Citation Format: John L. Sensintaffar, Allison Arnold, Craig Goodwin, Leah Hogdal, Subrata Shaw, James C. Tarr, Taekyu Lee, Edward Olejniczak, Stephen W. Fesik. Small molecule Mcl-1 inhibitors induce apoptosis and death in multiple cancer subtypes in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3727.
Abstract 3729: Novel radiation mitigators and anticancer drugs
Authors: Schiestl Robert H. , Rivina Yelena , Davoren Michael 
Published: July 2016

Abstract
The possibility of a radiation disaster from a nuclear detonation or accident has existed for over 50 years and spawned much of the basic research in radiobiology in the 1950-60s. The recent Fukushima accident was yet another reminder that there remains a dire need to develop novel therapies against radiation-induced toxicities. Here we report on the development of two novel radiation countermeasure therapies: CJ010 and Yel002. These small, biologically active, drug-like molecules were uncovered in the DEL high throughput assay reducing radiation-induced cyto- and geno-toxicity in yeast. Radiation-modulating activity was further confirmed in yeast plate-based DEL Assay: addition of either CJ010 or Yel002 to irradiated cultures reduced cell death and genomic instability. Further, the compounds increases survival to 75% in vivo following an LD100/30 dose of ionizing radiation (IR) with the first therapeutic injection administered 24 hours post exposure followed by injections at 48,72,96, and 120 hours. Additionally, treatment with Yel001 an analog of CJ010 and Yel002 compounds reduces radiation-induced leukemia from 90% to to 50% and 40% respectively. Of note, treatment with either Yel001 or Yel002 reduced spontaneous leukemia rate from 10% to 0%. Treatment with Yel002 following IR accelerates the recovery of the hematopoietic cells after sub-lethal exposures. In addition, treatment with Yel002 reduces EMS, MMS, UV, radioactive iodine, cigarette smoke extract as well as nitrogen mustard induced toxicity as well as genotoxicity showing a broad application spectrum. It also prolongs live of cells in a senescence assay. In addition Atm deficient mice live 16 weeks longer with weekly injection of Yel002 which is about 12 years in human life expectancy. In addition, Yel002 complements a zebrafish model of Diamond Blackfan Anemia. It works in yeast, CHO cells, different human cells, mice and zebrafish. Toxicity has not been observed in neither in vitro or in vivo administrations. Overall, Yel compounds have much potential as stockpile therapies for radiation-induced lethality and cancer: they are highly effective when administered up to 24hours post exposure, they reduce radiation-induce sequelae such as leukemia, and appear to have an acceptable toxicity profile.
Citation Format: Robert H. Schiestl, Yelena Rivina, Michael Davoren. Novel radiation mitigators and anticancer drugs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3729.
Abstract 3732: Combined targeting of NOXA and GSTpi effectively kills mantle cell lymphoma cells
Authors: Kleih Markus , Heine Simon , Dengler Michael , Schaaf Lea , Hoering Elisabeth , Horn Heike , Ott German , Aulitzky Walter E. , van der Kuip Heiko 
Published: July 2016

Abstract
Mantle cell lymphoma (MCL) is clinically characterized by an aggressive course and only transient response to chemotherapy. Therefore, new treatment strategies for this malignancy are warranted. The genetic hallmark of MCL is the t(11;14)(q13;q32) translocation leading to deregulated expression of cyclin D1. It has been reported recently that cyclin D1 overexpression in MCL is associated with constitutively high levels of GSTP1 and PMAIP1 transcripts, coding for the glutathion S-transferase pi (GSTpi) and for the proapoptotic protein NOXA, respectively. Whereas GSTpi protein is stably expressed, NOXA half-life turned out to be extremely short in this B cell lymphoma leading to constitutively low levels of this BH3-only protein in these cells. We asked if this MCL typical phenotype can be utilized for a selective treatment by stabilizing NOXA protein and concurrent inhibition of GSTpi activity. By screening different compounds known to stabilize NOXA protein and GSTpi inhibitors we found that combination of the fatty acid synthase (FASN) inhibitor Cerulenin together with Ezatiostat or Ethacrynic Acid is not only effective but also selective for MCL cells. Cell death induced by lethal doses of Cerulenin was dependent on Cyclin D1 since siRNA-mediated knockdown of Cyclin D1 partially rescued cells from apoptosis. Combinatory treatment of cells with sub-lethal doses of Cerulenin together with Ezatiostat/Ethacrynic Acid had no effect on viability of fibroblasts from normal donors but effectively killed both MCL cell lines as well as primary cells from MCL patients. Importantly, Cerulenin-mediated NOXA accumulation was significantly further elevated upon combination with GSTpi inhibitors and associated with a concomitant induction of reactive oxygen species (ROS) levels. Cell death was dependent on both NOXA and ROS since either inhibition of ROS by GSH or Catalase or siRNA-mediated knockdown of NOXA was sufficient to almost completely rescue cells from Cerulenin/GSTpi inhibitor induced apoptosis.
In conclusion we demonstrate that combined pharmacological inhibition of GSTpi activity and stabilization of NOXA protein via inhibition of fatty acid synthase might be an effective strategy to selectively kill MCL cells and offer novel treatment options.
Citation Format: Markus Kleih, Simon Heine, Michael Dengler, Lea Schaaf, Elisabeth Hoering, Heike Horn, German Ott, Walter E. Aulitzky, Heiko van der Kuip. Combined targeting of NOXA and GSTpi effectively kills mantle cell lymphoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3732.