bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3301: Guanidinium antisense oligonucleotides for cancer therapy.
Authors: Thomas Sufi M. , Ly Danith
Published: August 2014

Molecular targeted therapies are being tested in several clinical settings to target cancer. Most of the drugs being tested are small molecule inhibitors or antibodies that are specific to target proteins. Several of which have reduced target specificity or the tumor develops resistance to the drugs. This results in poor clinical outcomes. An approach that is highly specific and yet malleable enough to circumvent resistance mechanisms would be highly appealing. We have developed an antisense approach that can specifically bind to the mRNA of target proteins preventing translation and resulting in lower protein levels. In addition, unlike conventional antisense approaches, our antisense oligonucleotide carries a pseudo-peptide backbone with positively charged guanidinium residues rendering these resistant to nucleases and proteases. The oligomer binds nucleic acids with high sequence specificity and the hybridized complex is highly stable. As a proof of principle, we developed a 16 base antisense oligomer targeting the epidermal growth factor receptor (EGFR). EGFR is upregulated in several cancers and correlates with a poor prognosis. We tested the guanidinium-peptide nucleic acid (GPNA) EGFR antisense oligomer in tumors of the head and neck, lung and bladder. We carried out in vitro and in vivo studies including uptake and antitumor efficacy. Our data demonstrate that EGFR levels and cell survival are significantly reduced upon EGFRAS GPNA administration but not a scrambled control. Further, the EGFRAS GPNA effectively reduced levels of a constitutively active mutant EGFR (EGFRvIII). In vivo, EGFRAS GPNA had antitumor effects comparable to EGFR inhibitors that are under clinical testing namely erlotinib and cetuximab. Our data demonstrate that GPNA oligomers are an effective therapeutic approach against several tumor types.

Citation Format: Sufi M. Thomas, Danith Ly. Guanidinium antisense oligonucleotides for cancer therapy. [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 3301. doi:10.1158/1538-7445.AM2013-3301
Abstract 3302: Antisense oligonucleotide depletion of the mitotic kinesin Eg5 by direct delivery to the brain could be a useful strategy for treating glioma tumors.
Authors: Krona Cecilia C. , Boubaker Jihane , Friedmann-Morvinski Dinorah , Wong Alex , McAlonis-Downes Melissa , Koller Erich , Kim Aneeza S. , Hung Gene , Rigo Frank , Chun Seung , Vitre Benjamin , Bennett Frank , Verma Inder , Cleveland Don W.
Published: August 2014

To date, all approved chemotherapeutic agents which target the mitotic cell division interfere with spindle microtubule dynamics, leading to mitotic arrest and apoptosis. While effective, these drugs are subject to resistance mechanisms and they are also associated with a variety of side effects, including neurotoxicity. Their use in treating nervous system tumors is therefore not warranted.

One strategy to target mitosis, without damaging microtubules in non-dividing neurons, would be to inhibit key mitotic components, such as the mitotic kinesin Eg5, which is required for establishing a normal bipolar mitotic spindle. We have shown that glioblastoma cells depleted for Eg5 arrest in the next mitosis. After a prolonged arrest, they may slip out and become multinucleated, which will likely prevent further successful divisions or they may go into apoptosis. Further, mitotic arrest and induction of apoptosis in Eg5 depleted glioblastoma cells occur independent of p53, Rb-signalling and the PI3K-pathway suggesting that Eg5 is a potential therapeutic target for glioblastoma patients with different underlying genetic abnormalities

We have also tested the clinical feasibility of using a cell cycle targeting antisense oligonucleotide based therapy delivered directly to the central nervous system (CNS) as a novel treatment for glioblastoma tumors.

This work has demonstrated that intraventricular administration of ASOs can efficiently target cells in the CNS and be delivered to glioma-initiating neural stem cells transplanted into the cortex of naïve mice as well as to glioblastoma tumors in a genetically predisposed mouse model. This strategy is therefore a potential route of administration for treating glioblastoma tumors which originate in the CNS. Direct targeting of mitotic components in the brain should have a limited toxicity to non-cycling neurons and as a benefit, as long as the blood-brain barrier is intact direct CNS delivery should have minimal dose-limiting toxicity outside of the CNS. Ongoing studies will determine the effect of Eg5 inhibition on glioblastoma growth in vivo.

Citation Format: Cecilia C. Krona, Jihane Boubaker, Dinorah Friedmann-Morvinski, Alex Wong, Melissa McAlonis-Downes, Erich Koller, Aneeza S. Kim, Gene Hung, Frank Rigo, Seung Chun, Benjamin Vitre, Frank Bennett, Inder Verma, Don W. Cleveland. Antisense oligonucleotide depletion of the mitotic kinesin Eg5 by direct delivery to the brain could be a useful strategy for treating glioma tumors. [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 3302. doi:10.1158/1538-7445.AM2013-3302
Abstract 3309: T cell lymphoblastic leukemia in survivin TCR transgenic mice.
Authors: Cui Yongzhi , Onozawa Masahiro , Qin Haiying , Fry Terry , Aplan Peter , Mackall Crystal L.
Published: August 2014

Survivin has been considered a potential tumor antigen due to high expression in most cancers and limited expression in normal tissues. To explore whether survivin reactive TCRs mediate antitumor effects in mice, we generated TCR transgenic (Tg) mice with specificity for the H-2b restricted immunodominant epitope of survivin. Survivin reactive T cells were CD8+ and mediated immune reactivity toward survivin peptide pulsed targets. Some antitumor reactivity was observed, but it was not potent, and the survivin TCR Tg+ cells did not mediate objective tumor regression of survivin bearing tumors. Surprisingly, spontaneous T cell acute lymphoblastic leukemia (T-ALL) was observed beginning at 4-6 months of age in Tg+Rag+/- and Tg+Rag-/- mice. By one year of age, all survivin Tg+ mice succumbed to T-cell ALL. The leukemic cells were CD3+, survivin TCR+, and CD8+ or CD4-/CD8-Occurrence in 3 founders suggests that the transgene itself, rather than insertional mutagenesis, is causative. Given that survivin is expressed in the thymus, we postulated that the TCR-Tg serves as an oncogene via recognition of survivin peptides, which expands early thymic progenitors. In support of this, premalignant survivin TCR Tg+ thymi show expanded CD4-CD8-CD44-CD25- thymocytes and increased BrdU incorporation and leukemia incidence was significantly diminished TCR-Tg+β2m-/- mice. Subsequent to expansion of early thymic progenitors in response to TCR triggering, survivin TCR Tg+ cells acquire NOTCH mutations and upregulate CD25, implicating NOTCH signaling as a 2nd hit in this oncogenic process. At least one NOTCH1 mutation was found in all leukemias, with PEST (8/8), 5’ deletions (19/25) and heterodimerization mutations observed. T-ALL with NOTCH mutations was also observed at reduced frequencies, in TCR Tg mice with specificity to WT1 and gp100. We conclude that genetic engineering aimed at endowing hematopoietic or T lymphoid progenitors with the capacity to recognize tumor antigens expressed in the thymus could pose a risk for neoplasia, independent of insertional mutagenesis.

Citation Format: Yongzhi Cui, Masahiro Onozawa, Haiying Qin, Terry Fry, Peter Aplan, Crystal L. Mackall. T cell lymphoblastic leukemia in survivin TCR transgenic mice. [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 3309. doi:10.1158/1538-7445.AM2013-3309

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3310: Human artificial chromosome (HAC) vector with a conditional centromere for correction of human genetic deficiencies.
Authors: Kim Jung-Hyun , Kononenko Artem , Erliandri Indri , Kim Tae-Aug , Nakano Megumi , Iida Yuichi , Barrett J. Carl , Oshimura Mitsuo , Masumoto Hiroshi , Earnshaw William C. , Larionov Vladimir , Kouprina Natalay
Published: August 2014

Human artificial chromosome (HAC)-based vectors offer a promising system for delivery and expression of full-length human genes of any size. Due to their unique maintenance features and unlimited cloning capacity, HACs avoid the lack of copy number control and insertional mutagenesis caused by integration into host chromosomes that plague viral vectors. We previously described a synthetic HAC that can be easily eliminated from cell populations by inactivation of its conditional kinetochore. Here, we demonstrate the utility of this HAC, which has a unique gene acceptor site, for delivery of full-length genes and correction of genetic deficiencies in human cells. A battery of functional tests was performed to demonstrate expression of NBS1 and VHL genes from the HAC at physiological levels. We also show that phenotypes arising from stable gene expression can be reversed when cells are “cured” of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This generation of human artificial chromosomes should be suitable for studies of gene function and therapeutic applications.

Citation Format: Jung-Hyun Kim, Artem Kononenko, Indri Erliandri, Tae-Aug Kim, Megumi Nakano, Yuichi Iida, J. Carl Barrett, Mitsuo Oshimura, Hiroshi Masumoto, William C. Earnshaw, Vladimir Larionov, Natalay Kouprina. Human artificial chromosome (HAC) vector with a conditional centromere for correction of human genetic deficiencies. [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 3310. doi:10.1158/1538-7445.AM2013-3310
Abstract 3311: In vivo selection of S. typhimurium A1-R enhanced tumor-targeting variants.
Authors: Uehara Fuminori , Zhang Yong , Tome Yasunori , Maehara Hiroki , Kanaya Fuminori , Hoffman Robert M. , Zhao Ming
Published: August 2014

The S. typhimurium A1-R mutant (A1-R), which is auxotrophic for leu-arg, and selected for high antitumor virulence, has been previously shown to be effective as monotherapy against human breast, prostate, pancreatic, and fibrosarcoma tumors that have been orthotopically implanted in nude mice. In the present study, we generated an enhanced tumor-targeting S. typhimurium variant using in vivo selection. Dual-color murine mammary tumor (MMT) cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm were injected (1 × 106 cells) in both flanks of 4-week-old female nude mice. Seven days after tumor implantation, A1-R bacteria were injected (1 × 107 cfu iv). Five days after injection, the tumors were harvested and minced. The bacteria which invaded the tumor were isolated and seeded on LB agar. After overnight incubation, colonies were picked and purified and termed A1-R1. The procedure was repeated with A1-R1 to obtain A1-R2. To determine the efficacy of the new bacterial variants, MMT-dual cells were seeded onto the 6-well plates. After 48 hours incubation, A1-R, A1-R1, and A1-R2 (1 × 108 cfu/well) were added to each plate. After 0, 24, 48, 72, 96 hours incubation, each plate was observed by confocal microscopy. Compared with A1-R, the infection rate of cancer cells by A1-R1 and A1-R2 was increased. The A1-R1 and A1-R2 variant bacteria generated in vivo selection show promise for improved bacterial targeting in vivo.

Citation Format: Fuminori Uehara, Yong Zhang, Yasunori Tome, Hiroki Maehara, Fuminori Kanaya, Robert M. Hoffman, Ming Zhao. In vivo selection of S. typhimurium A1-R enhanced tumor-targeting variants. [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 3311. doi:10.1158/1538-7445.AM2013-3311
Abstract 3317: Efficacy of Salmonella typhimurium A1-R treatment on human patient pancreatic tumorgrafts.
Authors: Zhao Ming , Katz Matthew H.G. , Fleming Jason B. , Suetsugu Atsushi , Zhang Yong , Maawy Ali , Chishima Takashi , Tanaka Kuniya , Bouvet Michael , Hiroshima Yukihiko , Endo Itaru , Hoffman Robert M.
Published: August 2014

We have previously developed the genetically-modified Salmonella typhimurium A1-R (A1-R) strain that selectively targets tumors and demonstrated its efficacy for various types of cancer cell lines in nude mouse models. The aim of this study is to determine the efficacy A1-R treatment for human-patient pancreatic tumorgrafts. Pancreatic-cancer-patient tumor specimens were initially established subcutaneously in NOD/SCID mice immediately after surgery. The patient tumors were then harvested from NOD/SCID mice and passed orthotopically in transgenic nude mice, expressing fluorescent protein in order for the tumors to acquire fluorescent stroma for imaging. After confirmation of tumor growth by fluorescence imaging, the nude mice were treated in the following groups: (1) 5-fluorouracil (5FU) (10 mg/kg, ip); (2) cisplatin (CDDP) (10 mg/kg, ip); (3) gemcitabine (GEM) (150 mg/kg, ip); (4) A1-R (A1-R) (1.5x108 cfu/body, ip); and (5) PBS (vehicle/control) (ip). Control, 5FU, CDDP, GEM and A1-R injections were performed on a weekly basis from day-21 after tumor implantation for 4 weeks. Animals were sacrificed at 7 weeks, and tumors were harvested for analysis. Each treatment arm involved 5 tumor-bearing mice. No significant effects on body weight, morbidity, or severe toxicities were observed in any treatment arm. The tumor weight of each group was as follows: (1) 5FU, 0.044 ± 0.027g; (2) CDDP, 0.04 ± 0.032g; (3) GEM, 0.058 ± 0.051g; (4) A1-R, 0.106 ± 0.038g; and (5) PBS, 0.258 ± 0.209g. S. typhimurium A1-R treatment significantly reduced the tumor weight compared to control treatment (p = 0.011), as did the other treatments including 5FU (p = 0.005), CDDP (p = 0.004), and GEM (p = 0.001). S. typhimurium A1-R treatment was effective for human pancreatic tumorgrafts and could be a promising therapy for human pancreatic cancer.

Citation Format: Ming Zhao, Matthew H.G. Katz, Jason B. Fleming, Atsushi Suetsugu, Yong Zhang, Ali Maawy, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Yukihiko Hiroshima, Itaru Endo, Robert M. Hoffman. Efficacy of Salmonella typhimurium A1-R treatment on human patient pancreatic tumorgrafts. [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 3317. doi:10.1158/1538-7445.AM2013-3317
Abstract 3318: Cytotoxic effect of photosensitive fluorescent protein KillerRed-expressing adenovirus against human cancer cells.
Authors: Takehara Kiyoto , Tazawa Hiroshi , Hashimoto Yuuri , Kishimoto Hiroyuki , Narii Nobuhiro , Mizuguchi Hiroyuki , Fujiwara Toshiyoshi
Published: August 2014

Photodynamic therapy (PDT) is an anticancer therapy to induce tumor-specific cytotoxicity by accumulation of photosensitizers (PSs) and following non-toxic light irradiation to tumor tissues. Non-toxic light irradiation induces the PS-mediated generation of reactive oxygen species (ROS) and subsequent cytotoxicity in tumor cells. For clinical application, some kinds of PSs, such as photofrin and laserphyrin, are intravenously injected before light irradiation in anticancer PDT. However, the lack of sufficient and continuous accumulation of PSs in tumor tissues makes difficult to eradicate tumor cells by PDT. Therefore, the development of novel PSs that accumulate to tumor tissues more efficiently and persistently is required to improve the antitumor effect of PDT. A bioengineered fluorescent protein KillerRed has been previously reported to generate ROS upon green light irradiation, suggesting a potential of KillerRed fluorescent protein as a novel PS. We recently confirmed that transient or stable transfection with KillerRed-expressing plasmid vector efficiently suppressed the cell viability through induction of ROS-mediated apoptosis in various types of human cancer cells when combined with light irradiation. In this study, we constructed non-replicative adenovirus expressing cytotoxic KillerRed (Ad-KillerRed) or non-cytotoxic red fluorescent protein Katushka (Ad-Katushka). The cytotoxic effect of these viruses was investigated in human cancer H1299, HCT116 and HT29 cells in combination with light irradiation. Infection with Ad-KillerRed or Ad-Katushka did not induce any cytotoxic effect in human cancer cells without light irradiation. When combined with light irradiation, human cancer cells infected with Ad-KillerRed, but not Ad-Katushka, showed morphological change like round-shape. However, the cell viability was not significantly suppressed in Ad-KillerRed-infected cancer cells after light irradiation. These results suggest that replication-competent adenovirus is necessary to efficiently induce KillerRed-mediated cytotoxic effect in human cancer cells in combination with light irradiation. Now we are planning to generate the replication-competent oncolytic adenovirus expressing KillerRed and investigate its antitumor effect on the in vitro and in vivo settings.

Citation Format: Kiyoto Takehara, Hiroshi Tazawa, Yuuri Hashimoto, Hiroyuki Kishimoto, Nobuhiro Narii, Hiroyuki Mizuguchi, Toshiyoshi Fujiwara. Cytotoxic effect of photosensitive fluorescent protein KillerRed-expressing adenovirus against human cancer cells. [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 3318. doi:10.1158/1538-7445.AM2013-3318
Abstract 3319: Salmonella typhimurium A1-R inhibits aggressive pancreatic tumor growth and prolongs survival.
Authors: Maawy Ali , Zhang Yong , Bouvet Michael , Hoffman Robert M. , Zhao Ming
Published: August 2014

Salmonella typhimurium (A1-R) has been previously shown to target multiple tumor types. A1-R has been shown to prevent metastasis of the XPA-1 human pancreatic cancer cell line in nude mice. The aim of this study is the anti-tumor efficacy of A1-R against a more aggressive pancreatic cell line. Human MiaPaCa2 pancreatic cancer cells, expressing RFP, were cultured in vitro and subsequently infected with A1-R. Efficacy of infection was noted by intracellular bacteria with subsequent cell lysis and apoptosis. MiaPaCa2-RFP was implanted subcutaneously in nude mice. After tumor engraftment, the mice were subjected to intravenous therapy with A1-R (5×107 CFU) once a week for 3 weeks. The tumors were harvested and weighed after the treatment period. MiaPaCA2-RFP were subsequently surgically implanted orthotopically. Treatment was initiated 2 weeks later. Mice were treated with intravenous A1-R (5×107 CFU) once a week for 6 weeks, after which survival and tumor growth were recorded. In the subcutaneous tumor model, significant decreases in the rate of tumor growth (p = 0.049) and tumor size (p =0.004) were observed upon termination. In the orthotopic model, a significant decrease in the rate of tumor growth (p = 0.01) and increased survival was observed (p = 0.014). The mean survival of the treatment group and the control group was 49 days and 35 days, respectively. None of the mice showed any adverse events or weight loss from bacterial therapy. Further investigation will be expanded to other pancreatic cancer cell lines and human patient tumorgrafts in combination with chemotherapy with the goal of cure of pancreatic cancer.

Citation Format: Ali Maawy, Yong Zhang, Michael Bouvet, Robert M. Hoffman, Ming Zhao. Salmonella typhimurium A1-R inhibits aggressive pancreatic tumor growth and prolongs survival. [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 3319. doi:10.1158/1538-7445.AM2013-3319
Abstract 332: Establishment of animal model for metastasis of human lung cancers using NOJ/SCID mice.
Authors: Aragane Naoko , Sato Akemi , Kobayashi Naomi , Ide Masaru , Sueoka Eisaburo , Okada Seiji , Kimura Shinya
Published: August 2014

Lung cancer is the leading cause of cancer death among malignant tumors. Lymphogenic and distant metastases were found approximately 70% at the diagnosis, and most of recurrence after surgery occurs as distant metastasis. Therefore, it is important to establish the new strategy for control of metastatic lung cancers. Since metastasis consists of several steps such as loss of cell adhesion, intravasation, survival in circulation, exit into new tissues, and colonization in a distant site, a suitable animal model is indispensable for investigation of metastasis. In addition, recent investigations revealed that biological and molecular characteristics of cancer cells were different between primary and metastatic lesions, as well as among each metastatic site. To establish the animal model reflecting human metastatic lung cancers, we used NOD/Scid/Jak3null (NOJ) mice, which exhibit deficiencies in NK cell activity, macrophage and DC function, and complement activation as well as T and B cell deficiencies. This animal model enabled us to engraft various human cells more easily than previous immunodeficiency mouse models. At first, twenty kinds of human lung cancer cell lines were screened by expression pattern of E-cadherin and vimentin using western blot analysis, and two cell lines with high expression of vimentin and low expression of E-cadherin were selected. The cells (1×107/mouse) were injected into the dorsal flanks, and one cell line, H1975 showed lymphogenic metastasis to axillary and abdominal lymph nodes, resulting dissemination to pleura and diaphragm with ascites. Since H1975 cells carry epidermal growth factor receptor (EGFR) mutation, L858R and T790M, we can monitor the existence of cancer cells using a sensitive detection system, mutation-biased PCR and quenched probe system (MBP-QP), which were established in our laboratory. We have also tried to establish the animal model which lung cancer cells are inoculated into lung to mimic metastasis of human lung cancers. Using these model systems, we have been performing comprehensive analysis including mRNA expression, the mutation status of lung cancer related genes in each metastatic site, and compared the phosphorylation status of 38 kinds of molecules. Our goal of this study is to establish the animal model to reproduce phenotypes of metastasis of human lung cancers for screening of anti-metastatic agents.

Citation Format: Naoko Aragane, Akemi Sato, Naomi Kobayashi, Masaru Ide, Eisaburo Sueoka, Seiji Okada, Shinya Kimura. Establishment of animal model for metastasis of human lung cancers using NOJ/SCID mice. [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 332. doi:10.1158/1538-7445.AM2013-332
Abstract 3324: The discovery and optimisation of small-molecule inhibitors of human 5’-tyrosyl DNA phosphodiesterase (Tdp2).
Authors: Jordan Allan M. , Depledge Paul , Hamilton Nicola , Hitchin James , Hopkins Gemma , Maguire Laura , McGonagle Alison , Mould Daniel , Raoof Ali , Rushbrooke Mathew , Smith James , Smilth Kate , Thomson Graeme , Turlais Fabrice , Waddell Ian , Watson Mandy , Ogilvie Donald
Published: August 2014

Topoisomerases (topo) regulate DNA topology by the transient cleavage and re-ligation of DNA during transcription and replication. Topo II poisons such as etoposide can induce abortive DNA strand breaks in which topo II remains covalently bound to a 5’ DNA strand terminus via a phosphotyrosyl linker. Tyrosyl DNA phosphodiesterase 2 (Tdp2, TTRAP, EAPII) is a recently discovered human 5’-tyrosyl DNA phosphodiesterase which repairs this topo-mediated DNA damage, therefore playing a central role in maintaining normal DNA topology in cells. Cellular depletion of Tdp2 has been shown to result in an increased susceptibility and sensitivity to topo II-induced DNA double strand breaks. It has therefore been proposed that selective pharmacological inhibition of Tdp2 may be a novel approach to overcome intrinsic or acquired resistance to topo II targeted drug therapy.

To date, no known drug-like inhibitors of Tdp2 have been identified. We have recently reported a robust ‘mix and read’ HTS compatible assay and this was used to screen a diverse chemical library of approximately 92,000 compounds. From this endeavour, 2 distinct hit series have been identified. Following further chemical exploration of the original hit compounds, small molecule inhibitors of Tdp2 with sub-100nM potencies have been identified. This poster will describe our biological and chemical progress in this area, detailing SAR and some lessons learnt during investigation of this target.

Citation Format: Allan M. Jordan, Paul Depledge, Nicola Hamilton, James Hitchin, Gemma Hopkins, Laura Maguire, Alison McGonagle, Daniel Mould, Ali Raoof, Mathew Rushbrooke, James Smith, Kate Smilth, Graeme Thomson, Fabrice Turlais, Ian Waddell, Mandy Watson, Donald Ogilvie. The discovery and optimisation of small-molecule inhibitors of human 5’-tyrosyl DNA phosphodiesterase (Tdp2). [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 3324. doi:10.1158/1538-7445.AM2013-3324
Abstract 3326: TRAIL induces apoptosis preferentially in cell cycle arrested tumor cells, e.g., in tumor cells from children with acute lymphoblastic leukemia upon knockdown of cyclinE.
Authors: Ehrhardt Harald , Wachter Franziska , Grunert Michaela , Jeremias Irmela
Published: August 2014

Resting tumor cells represent a huge challenge during anti-cancer therapy due to their increased treatment resistance. Example given, during minimal residual disease remaining tumor cells might be non-cycling. TNF-related apoptosis-inducing ligand (TRAIL) is a putative future anti-cancer drug currently in phase I and II clinical studies. We recently showed that TRAIL is able to target leukemia stem cell surrogates (Blood 2012,119,4224). Here we tested the ability of TRAIL to target cell cycle arrested tumor cells.

Primary tumor cells from children with acute lymphoblastic leukemia were amplified in immuno-compromised mice and used for in vitro knockdown studies, besides tumor cell lines. Cell cycle arrest was induced in G0, G1 or G2 using cytotoxic drugs, phase-specific inhibitors or RNA interference against cyclinB and E. Cell cycle was visualized by flowcytometry analysis including phosphorylation of Serine 10 in Histone H3 to discriminate G2 from M phase.

Biochemical or molecular arrest at any point of the cell cycle increased TRAIL-induced apoptosis, independently from the tumor cell type, the cell cycle inhibitor used or the check point addressed. In contrast, when cell cycle arrest was disabled by addition of caffeine, the anti-tumor activity of TRAIL was reduced. Most importantly for clinical translation, tumor cells from three children with B-precursor or T-cell acute lymphoblastic leukemia showed increased TRAIL-induced apoptosis upon knockdown of either cyclinB or cyclinE arresting the cell cycle in G2 or G1, respectively.

Taken together and in contrast to most conventional cytotoxic drugs, TRAIL exerts enhanced anti-tumor activity against cell cycle arrested tumor cells. Therefore, TRAIL might represent an interesting drug to treat static tumor disease, e.g., during minimal residual disease.

Citation Format: Harald Ehrhardt, Franziska Wachter, Michaela Grunert, Irmela Jeremias. TRAIL induces apoptosis preferentially in cell cycle arrested tumor cells, e.g., in tumor cells from children with acute lymphoblastic leukemia upon knockdown of cyclinE. [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 3326. doi:10.1158/1538-7445.AM2013-3326

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3327: N-terminal domain of Tyrosyl-DNA phosphodiesterase I (Tdp1) is critical for its cellular function.
Authors: Cuya Selma M. , Wanzeck Keith C. , Comeaux Evan Q. , van Waardenburg Robert C.
Published: August 2014

Tyrosyl-DNA phosphodiesterase I (Tdp1) is a highly conserved eukaryotic DNA repair enzyme that catalyzes the resolution of 3’ and 5’ phospho-DNA adducts. Tdp1 has been implicated in the repair of DNA topoisomerase I (Top1)-DNA covalent complexes reversibly stabilized by camptothecins (CPTs) such as the FDA approved CPT derivatives topotecan and irinotecan. Tdp1 contains two HKD-motifs that provide two catalytic histidines that function as a nucleophile and an acid-base residue. A mutation of the acid-base His to Arg (H493R) in human Tdp1 is associated with the rare recessive ataxia SCAN1. hTdp1H493R and the analogous yeast mutant (Tdp1H432R) enhances cell sensitivity to CPT. In addition, the toxicity induced by this mutant is caused by the formation of a more stable Tdp1-DNA covalent intermediate, a rare characteristic for a DNA repair enzyme. However, this His to Arg substitution induces a minor toxic phenotype compared to other substitutions, such as the His432 to Asn substitution, which induces a Top1 dependent cellular lethality. A band depletion assay suggests that in vivo/cell Tdp1His432Asn remains in complex with Top1 on the DNA, which was not observed in a biochemical in vitro assay.

Biochemical studies revealed that Tdp1 catalysis is independent of the N-terminal domain. Among Tdp1 proteins, the N-terminal domain is poorly conserved in sequence and size (∼80aa for yeast and 140aa for human Tdp1).

We investigated the role of the N-terminal domain for Tdp1 activity in the cell. The N-terminal truncated proteins showed similar cellular distribution as the full-length proteins. Interestingly, the N-terminal truncated proteins did not display the toxicity that was observed with the full-length Tdp1 mutant proteins. This suggests that the N-terminal domain is a critical determinate of Tdp1 cellular function.

Preliminary results from our human cell line model shows similar results implying that the function of the N-terminal domain is conserved among Tdp1 proteins although it is poorly conserved. Further studies are necessary to ensure that these constructs are properly distributed. Moreover, the N-terminal domain of hTdp1 is post-translational modified, while our preliminary results suggest that this domain is important for protein-protein interaction and Tdp1 recruitment to its substrates. Understanding Tdp1 substrate and protein-interactions are important in the development of Tdp1 as therapeutic target.

This work is in part supported by the ADDA.

Citation Format: Selma M. Cuya, Keith C. Wanzeck, Evan Q. Comeaux, Robert C. van Waardenburg. N-terminal domain of Tyrosyl-DNA phosphodiesterase I (Tdp1) is critical for its cellular function. [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 3327. doi:10.1158/1538-7445.AM2013-3327
Abstract 3332: An artificial zinc finger glycosylase causes site-directed DNA damages and specifically kills the targeted cancer cells via synthetic lethality.
Authors: Fung Hua , Gerson Stanton L. , Barbas Carlos F.
Published: August 2014

Synthetic lethality is a new developed therapy, in which PARP inhibitor selectively kills BRCA-deficient cancer cells. However, BRCA-deficiency only accounts for a small fraction of cancers. We are interesting in applying synthetic lethal approach to target all cancers regardless BRCA statute. As single strand break (SSB) is the start point of the synthetic lethal, we hypothesized the targeted induction of persistent SSB at tumor unique sequences (cancer somatic mutation sequences) with assistance of synthetic lethality would specifically kill cancer cells. To test this principle we have constructed a six-zinc finger/mutant uracil glycosylase fusion protein (zinc finger glycosylase, ZFG). Upon binding to a specific 18-bp sequence by ZFG six-zinc binding domain, the mutated uracil glycosylase will remove a cytosine residue leaving an apyrimidinic site, which is subsequently convert to SSB by AP-endonuclease cleavage. The SSB lesion is capable of being repaired, however, upon repair, the zinc finger binding site is restored and the site-specific ZFG will again initiate a lesion leading to a persistent SSB. We have transfected ZFGs into BRCA2-deficient human breast cancer MCF7 and lung cancer A549 cells, the cells were then treated with PARP inhibitors. We observed increased killing of the ZFG-transfected cells compare to mock-transfected controls. Since BRCA2 deficiency causes homologous recombination (HR) repair defect, we next sought to determine if inhibitors of HR is a suitable substitute for loss of BRCA2. We are testing this concept by transfection of ZFG to BRCA2-proficient MCF7 cells with the treatment of combination of PARP and HR inhibitors, to determine whether 1. Combination of PARP and HR inhibitors treatments can achieve synthetic lethality; 2. ZFG constructs can enhance synthetic lethality. We conclude ZFG coupled with synthetic lethality is a promising direction towards the development of cancer specifically treatment.

Citation Format: Hua Fung, Stanton L. Gerson, Carlos F. Barbas. An artificial zinc finger glycosylase causes site-directed DNA damages and specifically kills the targeted cancer cells via synthetic lethality. [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 3332. doi:10.1158/1538-7445.AM2013-3332
Abstract 334: PIK3CA overexpression induces epithelial-mesenchymal transition and progression of head and neck squamous cell carcinonoma.
Authors: Du Li , Chen Xi , Shen Jingping , Zhang Fang , Lu Shi-Long
Published: August 2014

Head and neck squamous cell carcinoma (HNSCC) patients have a poor prognosis and invasion and metastasis are the major causes of mortality. However, the molecular mechanisms of HNSCC invasion and metastasis remain obscure. The phosphatidylinositol 3-kinase (PI3K) pathway regulates a wide range of cellular processes crucial for tumorigenesis. Amplification and mutation of PIK3CA, the gene coding for the catalytic subunit of PI3K, are among the most common genetic alterations in human HNSCC. To delineate the in vivo roles of PIK3CA during head and neck tumorigenesis, we developed a PIK3CA genetically engineered mouse model (GEMM), in which PIK3CA overexpression is specifically induced in the head and neck epithelia.

While overexpression of PIK3CA alone resulted in epithelial hyperplasia, and carcinoma in situ, it was not sufficient to induce visible tumor formation. In contrast, when we applied 4NQO, a DNA adduct-forming agent widely used as a tobacco surrogate together with PIK3CA overexpression, the PIK3CA-GEMM developed ∼50% poorly differentiated HNSCC compared to ∼10% of those in the control mice. PIK3CA tumors also had increased numbers of infiltrated leukocytes and angiogenesis. In addition, either regional lymph node metastases or lung metastases was observed in ∼40% of the PIK3CA-GEMM compared to no metastases in control mice. Characterizations of PIK3CA tumors revealed that PIK3CA tumors underwent epithelial and mesenchymal transition, and formed poorly differentiated metastatic tongue SCC in an orthotopical mouse model. Molecular analysis of the PIK3CA tumors suggests that rather than AKT, PDK1 facilitates progression of PI3K-driven HNSCC, and enhanced TGFbeta signaling with increased TGFbeta1 ligand and Smad3 may further contribute to this process. Lastly, overexpression of PIK3CA is associated with HNSCC progression and metastasis, and positively correlated with PDK1 expression but negatively with AKT activation, further verified the clinical relevance of our GEMM study. In summary, our results suggest that PIK3CA oncogene drives invasion and metastasis of HNSCC through PDK1 and TGFbeta signaling, and combined targeting of these pathways will reduce tumor progression in advanced HNSCC patients with PIK3CA alterations, which comprise about ∼40% of all HNSCC patients.

Citation Format: Li Du, Xi Chen, Jingping Shen, Fang Zhang, Shi-Long Lu. PIK3CA overexpression induces epithelial-mesenchymal transition and progression of head and neck squamous cell carcinonoma. [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 334. doi:10.1158/1538-7445.AM2013-334
Abstract 3348: Inhibition of MDMX by XI-011 induces p53-mediated apoptosis in head and neck cancer.
Authors: Ryu In Sun , Ryu Chang Hwan , Park Hyun Bae , Kim Eunhye , Roh Jong-Lyel
Published: August 2014

Overexpression of MDM2 or MDMX inactivates the tumor-suppressive function of p53. Restoration of p53 function counteracting these p53 repressors can lead to in vivo tumor regression. Therefore, we evaluated whether the restoration of p53 function by a small-molecule p53 activator XI-011, a pseudouria derivative NSC146109, induce apoptosis in head-and-neck cancer (HNC). The effects of XI-011 treatment in four HNC cell lines were tested, individually or in combination with nutlin-3a, an inhibitor of MDM2-p53 interactions, and its growth suppression, cell-cycle arrest, and apoptosis were assessed. XI-011 induced accumulation and reactivation of p53 in a wild-type TP53-bearing HNC cell line. XI-011 showed maximal growth suppression in tumor cells with overexpression of MDMX and MDMX-dependent p53 degradation. With down-regulation of MDMX mRNA and protein levels, XI-011 upregulated expression of p21 and proapoptotic genes and promoted apoptosis in a dose-dependent manner. Notably, the apoptotic response was blocked by inhibition of p53 or MDMX gene expression, demonstrating the p53 and MDMX dependence of XI-011 effects. XI-011 decreased the viability of HNC cells expression low levels of MDMX in a less-efficient manner. Interestingly, in combination therapy, XI-011 synergistically acted with nutlin-3a to inhibit growth of HNC cells. Our data suggest that the MDMX inhibition and p53 functional restoration by XI-011 effectively induced cytotoxicity and apoptosis in cancer cells, an action that may offer a promising strategy for treating HNC.

Citation Format: In Sun Ryu, Chang Hwan Ryu, Hyun Bae Park, Eunhye Kim, Jong-Lyel Roh. Inhibition of MDMX by XI-011 induces p53-mediated apoptosis in head and neck cancer. [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 3348. doi:10.1158/1538-7445.AM2013-3348
Abstract 3356: Comparative plasma and cerebrospinal (CSF) pharmacokinetics (PK) of vincristine sulfate standard preparation (VCR) and vincristine sulfate liposomes injection (VSLI) in a non-human primate (NHP) model.
Authors: Cole Diane E. , Lester-McCully Cynthia M. , Shah Nirali N. , Warren Katherine E. , Wayne Alan S. , Widemann Brigitte C.
Published: August 2014

Background: Vincristine has been used as standard therapy for adult and pediatric malignancies for 50 years. Cumulative neurotoxicity often requires capping the maximum single and cumulative VCR dose. VSLI is a novel preparation of VCR encapsulated in sphingomyelin/cholesterol liposomes. Following clinical trials that demonstrated safety, tolerability and efficacy in adults with relapsed/refractory acute lymphoblastic leukemia, VSLI has been granted FDA accelerated approval at a dose of 2.25 mg/m2given once weekly for that population. A pediatric Phase I trial is ongoing. We evaluated plasma and CSF PK of intravenously (IV) administered VCR and total VSLI (liposomal encapsulated and non-encapsulated) in our NHP model.

Methods: 0.1 mg/kg (1.2 mg/m2 human-equivalent dose) VCR over 1 minute or VSLI over 15 minutes was administered on separate occasions to each of 3 adult rhesus monkeys (Macaca mulatta). Serial paired plasma and CSF samples were collected for up to 48 hrs. Drug concentrations were quantified using HPLC/tandem mass spectrometry assays. For plasma samples, VCR standard curves were linear over a range of .0025-10.0 μg/ml (lower limit of quantification, .0025 μg/ml). Lower limit of detection for VCR in NHP CSF was .001 μg/ml. PK parameters were estimated using non-compartmental methods.

Results:

Plasma PK parameters (median and range) for VCR and VSLI Cmax (μg/ml) AUC0-infinity (μg*hr/ml) Cl (ml/min) Terminal T1/2 (hours) VCR 1.07 (0.99–2.1) 0.614 (0.336–0.676) 33.8 (29.6–54) 24.3 (14.2–54.4) VSLI 4.8 (3.7–7.3) 195 (163–312) 0.089 (0.073–0.113) 17.9 (13.9–21.5) Cmax = maximal concentration, AUC = area under the concentration x time curve, Cl = clearance, T1/2 = half life

VCR demonstrated a multi-exponential and VSLI a near-monoexponential plasma concentration versus time curve. The ratios ClVCR: ClVSLI for the individual NHP were 300, 463 and 477. Vincristine was not detected in any CSF sample.

Conclusions: The NHP model allowed for a direct comparison of VCR and VSLI PK. VCR Cl exceeds that of VSLI by approximately 400-fold. No penetration into the CSF was observed for either preparation.

Citation Format: Diane E. Cole, Cynthia M. Lester-McCully, Nirali N. Shah, Katherine E. Warren, Alan S. Wayne, Brigitte C. Widemann. Comparative plasma and cerebrospinal (CSF) pharmacokinetics (PK) of vincristine sulfate standard preparation (VCR) and vincristine sulfate liposomes injection (VSLI) in a non-human primate (NHP) model. [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 3356. doi:10.1158/1538-7445.AM2013-3356
Abstract 3365: Population pharmacokinetics of oxaliplatin: An analysis across human development in infants, children and adults.
Authors: Nikanjam Mina , Stewart Clinton F. , Takimoto Chris H. , Synold Timothy W. , Capparelli Edmund V.
Published: August 2014

Introduction: Oxaliplatin has been studied in both adult and pediatric clinical trials as therapy for a variety of solid and hematologic malignancies, but no comprehensive pharmacokinetic analysis has been conducted to define the impact of age on oxaliplatin disposition. We combined platinum ultrafiltrate concentration data from 4 separate studies to characterize oxaliplatin pharmacokinetics across a wide range of ages and renal function.

Methods: Oxaliplatin pharmacokinetic data from Phase I and II clinical trials included 2 pediatric studies (median age 11.2 yr, range 1.3 - 22.7) and 2 adult NCI organ dysfunction studies (median age 63 yr, range 28 - 86). Oxaliplatin was given as a 2 hour infusion; the majority of subjects received 130 mg/m2 (range 60 - 160 mg/m2). Plasma samples were obtained 2- 504 hours after dosing with the pharmacokinetic analysis performed using platinum ultrafiltrate concentrations (LLOQ 1-10 ng/mL). In total 161 subjects (1416 samples) were analyzed using nonlinear mixed effects modeling (NONMEM v. VI).

Results: Oxaliplatin concentration data required a four compartment model to characterize later samples. Serum creatinine (SCR) and age were independent predictors of clearance (CL) in the univariate screen, but age was not significant in the multivariate screen. CL increased with allometric weight, but decreased with SCR. Typical values were: 24.6 L/kg (volume of distribution) and 0.526 x (SCR/0.9)0.926 L/h/kg0.75 (CL, variability 63.8%). The final model was used to simulate a dose of 130 mg/m2 in 1000 subjects with the median (IQR) exposure (AUC), CL, and SCR as follows:

SCR (mg/dL) CL (L/hr/m2) AUC (mcg*hr/mL) Age 1 yr 0.7 (0.6–0.8) 7.4 (4.6–11.6) 17.5 (11.2–28.3) Age 5 yr 0.7 (0.6–0.8) 7.9 (4.9–11.7) 16.4 (11.1–26.3) Age 50 yr 1.0 (0.9–1.2) 6.4 (4.3–10.3) 20.3 (12.6–30.4) Age 50 yr (renal dysfunction) 2.2 (1.8–2.6) 3.2 (2.0–4.9) 40.5 (26.3–65.9)

Conclusions: This combined analysis suggests that earlier models limited to 2 or 3 compartments may not characterize the true terminal phase of oxaliplatin. Differences in age were entirely accounted for by weight and renal function. Standard BSA based dosing results in similar AUC and CL values between pediatric and adult patients with adequate renal function.

Citation Format: Mina Nikanjam, Clinton F. Stewart, Chris H. Takimoto, Timothy W. Synold, Edmund V. Capparelli. Population pharmacokinetics of oxaliplatin: An analysis across human development in infants, children and adults. [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 3365. doi:10.1158/1538-7445.AM2013-3365
Abstract 3368: Pre-clinical PK/PD/efficacy characterization of AZD1480, a JAK1/2 kinase inhibitor.
Authors: Schroeder Patricia
Published: August 2014

Constitutive activation of the transcription factor STAT3, manifest as Y-705 phosphorylation, is a feature of many hematologically-derived and solid tumor malignancies. Accumulating evidence indicates that JAK kinases are the key mediators of STAT activation in solid tumor models. AZD1480, an inhibitor of Janus kinases 1 and 2 (JAK1/2), is a bioavailable small molecule whose inhibition of the JAK-STAT signalling pathway may provide clinical benefit by suppression of the pleiotropic tumorigenic effects of constitutive STAT3 activation (tumor cell proliferation, motility and survival, tumor angiogenesis, tumor immune suppression).

Here we present a pre-clinical population-based effect-compartment pharmacokinetic / pharmacodynamic (PK/PD) model to describe the onset, intensity, and duration of pSTAT3 inhibition as a function of unbound concentration and time in the MDAH-2774 ovarian xenograft mouse model. A mathematical tumor growth inhibition model capturing the relationship between systemic exposure to AZD1480 and tumor growth inhibition was built to provide a pharmacokinetic rationale for tumor growth inhibition. Furthermore, the pSTAT3 inhibition PK/PD relationship for AZD1480 was quantitatively integrated into a tumor growth inhibition model providing a mechanistic pharmacodynamic rationale for tumor growth inhibition pre-clinically. Taken together, these pre-clinical data recommend that increasing inhibition of pSTAT3 correlates with greater tumor growth inhibition and provide a PK/PD/Efficacy platform of evidence for clinical hypothesis testing.

Citation Format: Patricia Schroeder. Pre-clinical PK/PD/efficacy characterization of AZD1480, a JAK1/2 kinase inhibitor. [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 3368. doi:10.1158/1538-7445.AM2013-3368

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.