bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3681: Nischarin disruption promotes breast tumor development in mouse models
Authors: Dong Shengli , Alahari Suresh K.
Published: July 2016

Nischarin is a novel tumor suppressor that was first discovered and characterized in our laboratory. The accumulating evidence suggests that Nischarin is a critical regulator of cell migration and breast tumor growth, metastasis and invasion. However, little is known about the exact function of Nischarin in in vivo physiological conditions, probably due to lack of any transgenic Nischarin animals. We recently generated Nischarin knockout mouse. This Nischarin knockout mouse has a dramatic unusual phenotype of mammary development and overall growth defect. Nischarin knockout mice do not spontaneously develop breast tumors. To determine the importance of Nischarin in regulation of breast cancer development, we bred Nischarin knockout virgin mice with PyMT (polyoma middle T antigen) and Neu background mice and the progeny produced tumors exclusively in the mammary gland. We found Nischarin knockout virgins with PyMT accelerated breast cancer onset and progression, which further suggests Nischarin inhibits tumor growth. Mammary epithelial cells (MECs) are surrounded by basement membrane, which mainly consists of extracellular matrix. We noted that the basement membrane of Nischarin knockout virgins contain much more collagen and fibronectin than those of wild type virgins. In addition, we observed that Nischarin disruption significantly changed extracellular acidification rates (ECAR) in mouse primary breast tumour cells. We suspected that this changed acidic microenvironment might facilitate breast tumor progression. Recent studies indicated that clinical anti-diabetic drug Metformin, an AMPK activator, inhibits breast cancer cell growth. Our data show that Het-PyMT and Null-PyMT primary cells were much more sensitive for metformin treatment. Thus we examined the importance of AMPK signaling in our mouse models. Interestingly, both tumor tissues and primary cells derived from Null-PyMT breast tumors had lower AMPK activity. Together, our data strongly support that Nischarin disruption promotes breast tumor development and AMPK signaling is important for Nischarin-mediated suppression of breast tumor.

Citation Format: Shengli Dong, Suresh K. Alahari. Nischarin disruption promotes breast tumor development in mouse models. [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 3681.
Abstract 3682: Phosphorylation mediated conformational changes defines nuclear role of phosphatase and tensin homology (PTEN) in tumor suppression
Authors: Malaney Prerna , Semidey-Hurtado Jonathan , Hardee Jamaal , Patel Deepal , Hennessey Daniel , Stanford Kate , Palumbo Emily , Tian Zhi , Allen-Gipson Diane , Davé. Vrushank
Published: July 2016

Loss of function of tumor suppressor PTEN (Phosphatase and tensin homolog) causes cancer in various tissues. PTEN C-terminal phosphorylation (pPTEN) inactivates PTEN, leading to multiple malignancies with increased severity. However, little is known about the molecular mechanisms underlying such inactivation. Therefore, the objective of our work is to ascertain the molecular mechanisms by which PTEN phosphorylation drives lung cancer. PTEN C-terminal phosphorylation at a serine-threonine cluster (Ser380, Thr382, Thr383 and Ser385) conformationally inactivates PTEN, abrogating its tumor suppressor function. Replacement of these serine/threonine residues with alanine generated an artificial phosphorylation-deficient mutant of PTEN (PTEN-4A), which is constitutively active. PTEN-4A suppressed cell proliferation and migration to a greater extent as compared to PTEN-WT. PTEN-4A preferentially localized to the nucleus and suppressed the E2F-mediated transcription of cell cycle genes. The nuclear localization sequence and phosphatase activity of PTEN-4A is critical for this transcriptional suppression. Immuno-precipitation assays show that PTEN physically associates with the transcription factor E2F1, a likely mechanism for its suppressive effect on E2F1 related genes. Further, deletion analysis of PTEN-4A protein revealed that the C2 domain is indispensable for suppression of E2F-related genes. Systematic transcriptional assays identify disease-associated C2 domain mutations that lose their ability to suppress E2F-mediated transcription, supporting the concept that these mutations are oncogenic in patients. Taken together, we reveal nuclear functions of PTEN-4A in tumor suppression that can be therapeutically leveraged for developing adjunctive cancer therapies. Small molecule inhibitors that hinder PTEN phosphorylation maybe utilized to activate PTEN nuclear function in tumors. Such adjunctive therapy has a high likelihood to reduce toxic doses of chemotherapeutic agents and targeted inhibitors, including kinase inhibitors that are being used in clinical settings.

Citation Format: Prerna Malaney, Jonathan Semidey-Hurtado, Jamaal Hardee, Deepal Patel, Daniel Hennessey, Kate Stanford, Emily Palumbo, Zhi Tian, Diane Allen-Gipson, Vrushank Davé. Phosphorylation mediated conformational changes defines nuclear role of phosphatase and tensin homology (PTEN) in tumor suppression. [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 3682.
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

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

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

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

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

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

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

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

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

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

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.
Abstract 3736: Schlafen 11 (SLFN11) blocks RNA synthesis upon replicative damage, a novel mechanism for killing cancer cells in response to DNA damaging agents
Authors: Murai Junko , Tang Sai-wen , Pommier Yves
Published: July 2016

The putative DNA/RNA helicase, SLFN11, is a recently discovered determinant of response to DNA damaging agents including topoisomerase inhibitors (camptothecin, irinotecan, topotecan, doxorubicin and etoposide) and cisplatin. SLFN11 is inactivated in approximately 40% of cancer cell lines (NCI-60 and CCLE). Until now, the molecular mechanisms of SLFN11 action in response to DNA damage have been unknown. Using isogenic SLFN11-knockout cells as well as SLFN11-overexpressing cells, we will show that SLFN11 irreversibly arrests cells in S-phase without cell cycle recovery under camptothecin treatment, whereas SLFN11-negative cells continue cell cycle progression. Notably, the action of SLFN11 is independent of ATR-mediated S-phase checkpoint. Co-immunostaining of 5-Ethynyl Uridine (EU) and gamma-H2AX revealed that SLFN11 blocks RNA synthesis upon replicative damage. Comprehensive analysis of cell cycle genes revealed that genes that drive S-phase progression are selectively turned-off in SLFN11-positive cells, whereas p53-dependent genes remain induced by camptothecin. Accordingly, critical proteins for S-phase progression such as cyclin A, cyclin B, CDK1 and CDK2 were suppressed in SLFN11-positive but not in SLFN11-negative cells, which causality links SLFN11-mediated S-phase arrest in camptothecin-treated cells with inactivation of cell cycle-driving pathways. This mechanism is supported by the fact that the CDK inhibitor roscovitine recapitulates the S-phase arrest even in SLFN11-deficient cells. Finally, by contrast to normal SLFN11, overexpression of SLFN11 helicase-dead mutant failed to block RNA synthesis and to kill camptothecin-treated cells, indicating that the helicase activity of SLFN11 is necessary for its RNA synthesis regulatory function. Our study reveals a novel cell cycle regulation mechanism by SLFN11 that links DNA damage and transcriptional responses in cancer cells with replicative DNA damage.

Citation Format: Junko Murai, Sai-wen Tang, Yves Pommier. Schlafen 11 (SLFN11) blocks RNA synthesis upon replicative damage, a novel mechanism for killing cancer cells in response to DNA damaging agents. [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 3736.
Abstract 3739: Efficacy of CD46-mediated Ad5/35 chimeric adenoviral gene therapy in colorectal cancers
Authors: Cho Young Seok , Do Hung Manh , Lee Sang-Jin , Hemmi Silvio , Joo Young-Eun , Jung Chaeyong
Published: July 2016

CD46 is a complement inhibitor membrane cofactor protein and also acts as a receptor for certain pathogenic microbes, including group B denovirus (Ad). Whereas many Ads infect cells through coxsackie-adenovirus receptor (CAR), CAR expression is downregulated in many cancers preventing effective therapeutics of Ad-based therapies. There have been increasing numbers of studies in cancer gene therapy by modification of Ad fiber knobs to utilize more ubiquitously expressed CD46. However, very limited information is available on expression status of CD46 in many cancers. Our tissue microarray data demonstrated that CD46 was generally overexpressed to cancers from prostate and colon. To seek the evidence whether colorectal cancers (CRC) are good target for Ad-mediated gene therapy, chimeric Ad5 vectors that are capable of targeting CD46 were employed. CD46-ovrexpressed cells showed significantly higher response not only to Ad5/35-GFP but to Ad5/35-TK/GCV suicide therapy. While CRC cells express variable levels of CD46, CD46 expression was positively correlated with Ad5/35-mediated GFP fluorescence and cell killing, demonstrating that HCT-116, DLD-1, and Caco-2 cells are highly responsive. Furthermore, injection of Ad5/35-TK/GCV caused significantlyhigher anti-cancer effects in nude mice bearing CD46-overexpressed cancer cells compared to mice with mock cells. Using 80 selected CRC, immunohistochemical studies showed that there is inverted correlation between CD46 expression and clinico-pathological parameters in terms of differentiation, invasion, metastasis, T stage, and survival, suggesting that adenoviral gene therapy may not be as effective to the patients with highly advanced colorectal cancers. Taken

altogether, our study demonstrated that CD46 is generally overexpressed in CRC in which group B-based adenoviral gene therapy seems to be suitable approach but careful consideration needs to be given to select cancer types and cancer status for effective cancer gene therapies.

Citation Format: Young Seok Cho, Hung Manh Do, Sang-Jin Lee, Silvio Hemmi, Young-Eun Joo, Chaeyong Jung. Efficacy of CD46-mediated Ad5/35 chimeric adenoviral gene therapy in colorectal cancers. [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 3739.
Abstract 3745: Tumor fibroblast targeting via uPAR retargeted measles virus: In vitro and in vivo effects
Authors: Jing Yuqi , Zaias Julia , Merchan Jaime
Published: July 2016

Tumor stromal cell components, in particular cancer associated fibroblasts, play an important role in cancer progression. Few studies have focused on stromal fibroblast targeting by oncolytic viruses. The urokinase receptor (uPAR) is a clinically and biologically validated target, which is overexpressed in tumor and stromal cells compared to non-cancer tissues. MV-h-uPA and MV-m-uPA are fully retargeted oncolytic measles viruses directed against human and murine uPAR, respectively, which have shown in vitro and in vivo safety and antitumor effects. Species specific retargeted viral vectors allow to dissect the specific effects of the viruses on murine vs. human tissues in xenograft models. Our aim is to characterize the in vitro and in vivo effects of stromal targeting by oncolytic measles virus via uPAR, with a focus on tumor fibroblasts. In vitro, MV-h-uPA and MV-m-uPA preferentially infected and induced cytotoxicity in human cancer associated fibroblasts (CAF 19, CAF 23) as well as murine fibroblasts (3T3), compared to non-tumorigenic fibroblasts. Murine-murine and human-human fibroblast to cancer cell viral transfer via heterofusion was observed after fibroblast infection by species specific MV-uPA, in breast, colon and renal cancer models, while no viral transfer was observed between cells of different species. In vivo, systemic administration of the murine uPA retargeted virus (MV-m-uPA) significantly decreased tumor progression and prolonged survival in a human breast cancer xenograft model (MDA-MD231), where the host stroma expresses murine uPAR (target of MV-m-uPA). Tumor studies revealed induction of apoptosis (TUNEL assay), while no significant effects on cancer cell proliferation was observed. Dual staining for measles virus nucleocapsid proteins and fibroblasts markers demonstrated viral infection of fibroblasts in treated tumors. Gene expression studies using murine as well as human specific arrays were performed to characterize the effects of stromal targeting by the murine retargeted virus on murine stroma as well as the indirect effects on human cancer cells in vivo.

In conclusion, for the first time our results show feasibility and antitumor effects of stromal fibroblast targeting by oncolytic measles virus via uPAR and demonstrate that stromal fibroblasts are viable targets for oncolytic virotherapy. Studies characterizing other stromal cell components and evaluation of the molecular changes in the tumor stroma as result of viral infection are underway.

Citation Format: Yuqi Jing, Julia Zaias, Jaime Merchan. Tumor fibroblast targeting via uPAR retargeted measles virus: In vitro and in vivo effects. [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 3745.
Abstract 3753: Improved oncolytic virotherapy by increasing adenovirus spread
Authors: Wechman Stephen L. , Rao Xiao-Mei , McMasters Kelly Marc , Zhou Heshan Sam
Published: July 2016

Lung cancer is the leading cause of cancer-related death worldwide and kills more patients than the next four cancers combined (Colon, breast, prostate, and pancreas). Currently available lung cancer treatments have not been effective. Therefore the development of novel lung cancer therapeutics is of critical importance. One emerging treatment option is the use of E1b-deleted adenoviruses (Ads) which preferentially lyse cancer cells. Although E1b-deleted Ads have been applied in clinical trials, their efficacy remains low. We have developed a novel oncolytic vector, AdUV, via the bioselection of the cancer selective E1b-deleted Ad Adhz60. We have demonstrated that AdUV is much more potent against multiple cancer cell lines than the parental vector Adhz60 in vitro and suppressed tumor growth in vivo.

We compared AdUV treated cells to cells treated with the wildtype Ad5 and the E1b-deleted Adhz60. AdUV lysed A549 lung cancer cells more efficiently than Adhz60, but neither AdUV nor Adhz60 killed MRC5 and HBEC normal lung cells. Possible mechanisms of action were then investigated to determine how AdUV lysed A549 cells more effectively. AdUV virions were shown to release from infected A549 cells and induce autophagy more effectively than both Adhz60 and Ad5. Autophagy induction is known to support Ad replication and spread through cancer cell monolayers. The plaque assay was used to verify if AdUV could spread more effectively than Ad5 and Adhz60 because viruses which efficiently replicate and release from cells are known to form larger plaques. The average size of plaques formed by AdUV is 340% and 214% larger than those plaques formed by Adhz60 and Ad5, respectively. In vivo, AdUV was non-lethal to athymic nude mice and strongly suppressed A549-xenograft tumor growth, prolonging murine survival. These results indicate that AdUV is a more effective, safe oncolytic Ad vector. Our findings also demonstrate the utility of bioselection of the generation of more effective cancer therapeutic vectors.

Citation Format: Stephen L. Wechman, Xiao-Mei Rao, Kelly Marc McMasters, Heshan Sam Zhou. Improved oncolytic virotherapy by increasing adenovirus spread. [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 3753.
Abstract 3757: A novel class of chemicals that react with damaged DNA and specifically kill B-cell cancers
Authors: Wei Shanqiao , Watuthanthrige Madusha , Shalhout Sophia , Bhagwat Ashok S.
Published: July 2016

Majority of B-cell malignancies arise in germinal center, where the processes of somatic hypermuation (SHM) and class switch recombination (CSR) for antibody maturation are initiated by activation-induced demainase (AID). AID belongs to the AID/APOBEC family and deaminates cytosine in DNA creating uracil. We have previously shown that B-cell lymphoma cell lines and patient tumors express AID at high levels and contain high levels of uracils in their genome. These cells also contain uracil-DNA glycosylase that removes uracils generating high levels of abasic (AP) sites. AP sites exist in equilibrium with the open form of deoxyribose sugar that reacts with alkoxyamines forming a stable oxime. This principle was used to block the repair of excess AP sites in B cell cancers using an alkoxyamine, AA3. We found that AA3 effectively kills B-cell lymphoma cell lines which have high levels of endogenous AP sites. In contrast, AA3 is not toxic to normal human B cells and mouse splenocytes, as well as non-hematological cancers. To elucidate the molecular mechanism of selective cell killing by AA3, we compared the number of AP sites in B cell cancers and non-hematological cancers and confirmed that B cell cancer cells contained many more AP sites in their genome. Additionally, we found that AA3 was covalently linked to B cell cancer genome and AA3-DNA adducts caused cell death by blocking DNA replication and elevating DNA strand breaks. To determine which functionality of AA3 is responsible for its cytotoxicity and to discover better candidates for killing B cell cancers, we designed and synthesized a series of AA3 analogues. Using this approach, we have defined the functional groups required for AA3 toxicity. Overall, we have discovered that AA3 is selectively toxic to B cell cancers. This new family of chemicals could be developed further as novel and unique anti-cancer drugs.

Citation Format: Shanqiao Wei, Madusha Watuthanthrige, Sophia Shalhout, Ashok S. Bhagwat. A novel class of chemicals that react with damaged DNA and specifically kill B-cell cancers. [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 3757.
Abstract 3765: Induction of amineoxidases as a possible mechanism of action of tamoxifen metabolites
Authors: Thomas T. J. , Keinanen Tuomo A. , Hyvonen Mervi T.
Published: July 2016

To elucidate the mechanism of action of endoxifen in breast cancer, we determined its effects on estradiol-mediated growth of ERalpha-positive MCF-7 cells. Endoxifen is the active metabolite of tamoxifen; the most widely used selective estrogen receptor modulator for anti-estrogen therapy for women with estrogen receptor alpha-positive breast cancer. Clinical trials recognized that tamoxifen was a prodrug and got converted to active metabolites in the human body. Earlier studies recognized 4-hydroxytamoxifen (4HT) as the active metabolite of tamoxifen, although recent studies showed that the concentration of endoxifen was up to 10-fold higher than that of 4HT in patients treated with tamoxifen. These metabolites are formed by the action of cytochrome P450 2D6 (CYP2D6). We used quantitative PCR analysis to examine the effects of endoxifen on estrogenic stimulation of early response genes, c-myc, c-fos and Tff1. Since the natural polyamines, putrescine, spermidine and spermine, play important roles in estrogenic function, we also measured the activities of enzymes in the polyamine biosynthetic/metabolic pathway: ornithine decarboxylase (ODC), S-adenosyl-L-methionine decarboxylase (AdoMetDC), spermidine/spermine N1-acetyltransferase (SSAT), spermine oxidase (SMO), and acetylpolyamine oxidase (APAO). Polyamine levels were quantified by high performance liquid chromatography. Our results showed that estradiol increased the proliferation of MCF-7 cells by 2- to 3-fold and endoxifen suppressed the effects of estradiol on cell growth. The expression of c-myc, c-fos and Tff1 genes was significantly increased by estradiol, while the addition of endoxifen suppressed estradiol-mediated increase in gene expression. Estradiol increased the activity of ODC and AdoMetDC, whereas endoxifen suppressed the activity of these enzymes. Endoxifen had no effect on SSAT activity, whereas it significantly increased activities of amineoxidases, SMO and APAO. Estradiol increased putrescine and spermidine levels and endoxifen inhibited this increase. There was no significant effect on spermine levels. These data suggest that endoxifen exerts its growth inhibitory effects on MCF-7 cells by suppressing the expression of estradiol-stimulated oncogenes and polyamine biosynthetic pathway. The antiestrogenic effect of endoxifen is also manifested through the stimulation of polyamine oxidase enzymes, SMO and APAO. These results indicate for the first time that the upregulation of SMO and APAO is important in the mechanism of action of endoxifen in breast cancer cells.

Citation Format: T. J. Thomas, Tuomo A. Keinanen, Mervi T. Hyvonen. Induction of amineoxidases as a possible mechanism of action of tamoxifen metabolites. [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 3765.
Abstract 3766: Targeting promoter regions of c-Myc and Bcl-2 in AML cells using G-quadruplex interacting drugs
Authors: Turnidge Megan , Montoya Justin J. , Patel Apurvi , Lee David W. , Aleem Eiman , Wai Daniel H. , Hurley Laurence H. , Gokhale Vijay , Arceci Robert J. , Azorsa David O.
Published: July 2016

Cancer is a disease that can be characterized by overexpression of key oncogenic drivers that support tumor development and maintenance. In many instances, these oncogenic drivers are ‘undruggable’ because of structural challenges, the inability to effectively inhibit high concentrations of overexpressed proteins, and the development of drug resistance mutations. An alternative therapeutic approach is to directly inhibit gene transcription by targeting unique secondary DNA structures, called G-quadruplexes, that are associated with subsets of promoters. We investigated the activity of a class of compounds termed G-quadruplex Interacting Drugs (GQIDs) that are able to shut down the expression of specific oncogenic drivers, such as c-Myc and Bcl-2, used by tumor cells for growth and survival. We tested the activity of two GQIDs, GQC-05 and GSA-1103, on cell growth of a panel of eight pediatric and eight adult acute myeloid leukemia (AML) cell lines. Drug dose response analysis showed IC50 values ranging from approximately 10 nM to 1 μM for GQC-05 and from 40 nM to 2 μM for GSA-1103. The AML cell lines had different sensitivities to each GQID indicating a different mechanism of action for each compound. Three AML cell lines that were highly resistant to cytarabine were among the more sensitive to GQC-05. Four cell lines sensitive to GQC-05 had high expression of either c-Myc and/or Bcl-2, both of which are potential targets of these two GQIDs. Furthermore, treatment of the highly sensitive line MV-4-11 with GQC-05 showed a 3-4 fold decrease in expression of c-Myc and Bcl-2 mRNA. Furthermore, GQC-05 treatment resulted in decreased levels of both c-Myc and Bcl-2 proteins. These studies will help define a novel approach of repressing key drivers of leukemia by targeting selective promoter structures.

Citation Format: Megan Turnidge, Justin J. Montoya, Apurvi Patel, David W. Lee, Eiman Aleem, Daniel H. Wai, Laurence H. Hurley, Vijay Gokhale, Robert J. Arceci, David O. Azorsa. Targeting promoter regions of c-Myc and Bcl-2 in AML cells using G-quadruplex interacting 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 3766.