bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3266: Repurposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints.
Authors: Beeharry Neil , Fink Lauren , Banina Eugenia , Khazak Vladimir , Skobeleva Natalia , Peterson Jeffrey R. , Astsaturov Igor , Yen Timothy J.
Published: August 2014

Many conventional chemotherapies act as DNA damaging agents. Chemotherapy activates cell cycle checkpoint signaling to prevent cells from entering mitosis with either damaged or unreplicated DNA. Based on the observations that interference with the checkpoint mechanism (checkpoint override) results in cell death via mitotic catastrophe, the development of checkpoint inhibitors is actively being pursued as an anti-cancer therapy. In a cell-based screen of clinically relevant kinase inhibitors we identified three compounds that display checkpoint override properties: isomer of bosutinib, a recently FDA-approved Abl and Src inhibitor; BEZ235, a PI3K/mTOR inhibitor; and dovitinib, a multi-receptor tyrosine kinase inhibitor. Most interestingly, all compounds display off-target activity towards DNA damage checkpoint kinases including Chk1, ATR and Wee1. We determined these kinase inhibitors potently sensitized a panel of pancreatic cancer cell lines to S-phase acting (Gemcitabine, cisplatin) or G2-phase arresting (doxorubicin) DNA-damaging agents. Using a combination of live-cell videomicroscopy and immunofluorescence, we demonstrated that the inhibitors forced drug-treated cells to override the DNA damage induced checkpoint activation and aberrantly enter mitosis. The aberrant mitoses had characteristic chromatin fragmentation and that kinetochores and centromeres appeared to be detached from the chromatin mass, in a manner reminiscent of mitosis with unreplicated genomes (MUGs). As a result, cells died either in mitosis or due to mitotic catastrophe. The efficacy of pulsatile (24h and 48h after chemotherapy) administration of these previously unsuspected checkpoint inhibitors was further validated in tumor explants and cells isolated directly from a pancreatic cancer patient who is currently undergoing treatment. Tellingly, xenograft studies showed that gemcitabine and bosutinib isomer had significant activity as single agents but the combination significantly prevented tumor growth, even after drug removal. Our findings suggest that kinase inhibitors targeting cell cycle checkpoints, albeit in an off-target manner, may be fast-tracked into the clinic to enhance the efficacy of standard chemotherapies.

Citation Format: Neil Beeharry, Lauren Fink, Eugenia Banina, Vladimir Khazak, Natalia Skobeleva, Jeffrey R. Peterson, Igor Astsaturov, Timothy J. Yen. Repurposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints. [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 3266. doi:10.1158/1538-7445.AM2013-3266
Abstract 3285: Synergistic anticancer effects of resveratrol and clofarabine in malignant mesothelioma cells.
Authors: Lee David M. , Lee Yoon-Jin , Im Jae-Hyuk , Won Seong-Youn , Nam Hae-Seon , Lee Sang-Han
Published: August 2014

Malignant mesothelioma is highly resistant to apoptosis. Hence, new strategies to enhance the apoptotic signal and overcome resistance to therapeutic drugs are necessary to improve the therapeutic efficacy of malignant mesothelioma. Here, we investigated the effects of the combined treatment of various phytochemicals as a chemo-potentiator in combination with the chemotherapeutic agent clofarabine in malignant mesothelioma MSTO-211H cells and normal mesothelial Met-5A cells. Treatment with resveratrol in combination with clofarabine at a subtoxic concentration produced a synergistic antiproliferative effect and induced apoptotic cell death, as evidenced by increased subG1 peak on flow cytometric analysis, chromatin condensation and nuclear fragmentation on DAPI staining, and DNA laddering on agarose gel electrophoresis, in MSTO-211H cells, but not in Met-5A cells. In MSTO-211H cells, endogenous expression of the transcription factors Nrf2 and Sp1 proteins, several Nrf2 and Sp1-regulated gene products (HO-1, c-Met, and cyclin D1), and Akt phosphorylation, were effectively suppressed by the combined treatment of clofarabine and resveratrol. Furthermore, the nuclear accumulations of Nrf2 and Sp1 were inhibited by the combination treatment. In combination with clofarabine, the ability of resveratrol to increase the contents of the phosphorylated p53 and the apoptotic molecules, PARP and cleaved caspase 3, was also evident in a time- and dose-dependent experiment. Taken together, the data provide evidence that the synergistic antiproliferative effect of resveratrol and clofarabine is linked to simultaneous suppression of multi-target molecules, such as Nrf2, Sp1, and Akt activities, and suggest that this combination may have therapeutic value in treatment of malignant mesothelioma.

Citation Format: David M. Lee, Yoon-Jin Lee, Jae-Hyuk Im, Seong-Youn Won, Hae-Seon Nam, Sang-Han Lee. Synergistic anticancer effects of resveratrol and clofarabine in malignant mesothelioma 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 3285. doi:10.1158/1538-7445.AM2013-3285
Abstract 3287: Sensitizing cancer cells to chemotherapy.
Authors: Wolak Kathryn E. , Bainer Russell , Skol Andrew , Onel Kenan
Published: August 2014

A wide range of cancers are either initially resistant to chemotherapy or are sensitive, but subsequently relapse through acquired drug resistance. Alterations in the p53 pathway are associated with drug resistance, underscoring its central role in determining response to chemotherapy. Using pediatric B-cell acute lymphoblastic leukemia as a model, we identified a set of p53-regulated transcriptional targets that comprise an apoptotic gene signature significantly associated with response to therapy and independently prognostic for outcome in pediatric ALL. We hypothesize that cells can be primed for an enhanced apoptotic response to chemotherapy by modulating expression levels of the p53-regulated apoptotic signature genes with small molecules before administering chemotherapy. We conducted a high-throughput screen to identify novel compounds that prime ALL cells for apoptosis to convert a chemotherapy-resistant cell into one that is sensitive. This screen was performed using 83,745 compounds in both the wild-type p53 cell line TK6 and the mutant-p53 line, WTK1. Cells were treated with either compound alone or compound plus daunorubicin, a drug used to treat ALL. Compounds were normalized and scored by using both the B-score and the robust Z-score. “Hits” were defined as compounds that do not significantly alter viability of the cells alone, but significantly increase daunorubicin-induced cell death. We confirmed the effects of the compounds in other B-cell ALL cell lines. In an effort in investigate mechanism, we looked at synergism using other chemotherapies with different modes of action. One compound was very synergistic with cisplatin, and we then extended the experiment to other cell types. We found that the synergism with cisplatin holds in all cell types tested, including T-cell ALL, lymphoma, breast, ovarian and colon cancer. In paired cisplatin-sensitive and resistant ovarian cancer cell lines, we find that the non-toxic doses of the compound can sensitize the resistant line and convert it to respond to cisplatin at the same doses as the parental sensitive line. Looking at the pathways induced by these compounds, we find important components in the cellular response to chemotherapy that can be manipulated to convert a chemoresistant cell into one that is sensitive.

Citation Format: Kathryn E. Wolak, Russell Bainer, Andrew Skol, Kenan Onel. Sensitizing cancer cells to chemotherapy. [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 3287. doi:10.1158/1538-7445.AM2013-3287
Abstract 3305: Sustained production of a soluble IGF-I receptor by gutless adenovirus - transduced host cells protects from tumor growth in the liver .
Authors: Wang Ni , Lu Yifan , Pilotte Amelie , Pinard Maxime , Gilbert Rénald , Massie Bernard , Brodt Pnina
Published: August 2014

The IGF-I receptor (IGF-IR) plays an important role in malignant disease by increasing cancer cell survival and promoting metastasis. It is presently the target of several drugs in clinical trials. Gene therapy is currently being developed for cancer treatment using different nanoparticle vectors designed to deliver therapeutic genes that could induce cell death, increase sensitivity to chemotherapy, block angiogenesis or enhance the anti-cancer immunological response. Previously, we have shown that the implantation of autologous bone-marrow stromal cells producing a soluble form of IGF-IR (sIGFIR) inhibited experimental liver metastasis of several tumor types in mice. Here we evaluated the utility of adenovirus-based gene delivery for generating therapeutically effective plasma levels of this decoy. A third generation gutless adenovirus was selected to deliver the sIGFIR gene because of its reduced immunogenicity and preferential tropism for the liver, the main source of endocrine IGF-1. We found that HEK-293 cells transduced by this, but not by control adenoviruses, secreted soluble receptor protein that blocked IGF-I-induced tumor cell migration, proliferation and survival in vitro. Following virus injection in vivo, viral DNA was detectable by PCR in several host organs, particularly the liver, and this resulted in the production of measurable sIGFIR plasma levels for up to 21 days post injection. In mice producing virus-encoded sIGFIR, experimental liver metastasis was inhibited, indicating that sIGFIR levels were therapeutically effective. The results show that adenovirus-based delivery of inhibitory soluble proteins can provide an effective strategy for blockade of cancer cell growth.

Citation Format: Ni Wang, Yifan Lu, Amelie Pilotte, Maxime Pinard, Rénald Gilbert, Bernard Massie, Pnina Brodt. Sustained production of a soluble IGF-I receptor by gutless adenovirus - transduced host cells protects from tumor growth in the liver . [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 3305. doi:10.1158/1538-7445.AM2013-3305
Abstract 3306: Traditional Chinese Medicine (TCM) enhances efficacy and reduces toxicity of tumor-targeting S. typhimurium A1-R in a syngeneic breast cancer metastatic mouse model.
Authors: Zhang Yong , Zhang Nan , Su Shibing , Hoffman Robert M. , Zhao Ming
Published: August 2014

We have previously developed a bacterial cancer therapy strategy using the genetically-engineered strain, Salmonella typhimurium A1-R (A1-R). A1-R has been shown to be effective against metastatic human prostate, breast, and pancreatic cancer cell lines as well as osteosarcoma, fibrosarcoma, and glioma cell lines in nude mouse models. In the current study, we have evaluated Traditional Chinese Medicine (TCM) (Huang Zi, Mai Men Dong, Mu Dan Pi, Sheng Di Huang) in combination with A1-R bacterial therapy in a syngeneic orthotropic model of 4T1 mouse breast cancer. TCM treatment improved the host's ability to clear bacteria from the liver and spleen and prevent body weight loss after bacterial infection. Fur discoloration and reduced activity was also observed in mice treated with A1-R only, but ameliorated when bacterial treatment was combined with TCM. The combination of TCM and A1-R enabled S. typhimurium to be safely administered at a dose of 1×107 CFU once a week i.v. for a long period of time in immunocompetent mice. Efficacy and survival were increased by combination treatment compared to monotherapy with A1-R (1 × 107CFU) . The results of the present study suggest that TCM potentiates A1-R cancer therapy.

Citation Format: Yong Zhang, Nan Zhang, Shibing Su, Robert M. Hoffman, Ming Zhao. Traditional Chinese Medicine (TCM) enhances efficacy and reduces toxicity of tumor-targeting S. typhimurium A1-R in a syngeneic breast cancer metastatic mouse 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 3306. doi:10.1158/1538-7445.AM2013-3306
Abstract 331: Head and neck and skin squamous cell carcinoma developed in a BRCA2 knockout mouse model.
Authors: Kamradt Michael , Zou Chaozhong , Liu Lin , Wang Yuexia , Fitchev Philip P. , Worman Howard J. , Crawford Susan E. , Talamonti Mark , Gao Qingshen
Published: August 2014

BRCA1 and BRCA2 were originally identified as breast cancer tumor suppressor genes. Germline mutations of BRCA1 have been found to predispose the carrier mainly to breast and ovarian cancers. However, germline mutations of BRCA2 can cause a much wider spectrum of cancers, including breast, ovarian, pancreatic and prostate cancers. There are conflicting reports on whether BRCA2 mutations also contribute to the tumorigenesis of head and neck squamous cell carcinoma (HNSCC). Previously, it had been reported that K14-cre; BRCA2f/f mice do not develop any cancer of epithelial origin even at 600 days of age in a 129/C57 background. Here, we generated a K14-cre mediated BRCA2 knockout mouse model in a different mouse strain background than previously reported. To our surprise, we found that K14-cre; BRCA2 f/f mice started to develop tumors at seven months of age. Most of the tumors were in the head and neck region, while others were skin tumors in other locations. The head and neck tumors showed no skin involvement or ulceration. Tumor growth in many mice eventually affected their food intake and they had to be euthanized for humane reason. At around 11 months of age, we sacrificed the remaining mice and subjected them to necroscopy. All the visually suspicious tissues were processed for pathologic examination. In total, we found 8 out of 31 K14-cre; BRCA2 f/f mice (26%) with pathologically confirmed head and neck cancer and 4 out 31 K14-cre; BRCA2 f/f mice (13%) with skin cancer. None of the 28 control BRCA2 f/f mice developed any cancer at this age. The head and neck cancers are squamous cell carcinomas arising from the mucosa surface and form large, palpable masses. In some areas, the tumors are well-differentiated with evidence of abundant keratin production, while in other areas they are less differentiated and exhibit high nuclear to cytoplasmic ratios, nuclear aytpia, mitoses and central necrosis. These histological features are highly reminiscent of the pathological characteristics of human HNSCC. This finding strongly indicates that mutations of BRCA2 play an important role in the tumorigenesis of HNSCC in a specific genetic background.

Citation Format: Michael Kamradt, Chaozhong Zou, Lin Liu, Yuexia Wang, Philip P. Fitchev, Howard J. Worman, Susan E. Crawford, Mark Talamonti, Qingshen Gao. Head and neck and skin squamous cell carcinoma developed in a BRCA2 knockout mouse 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 331. doi:10.1158/1538-7445.AM2013-331
Abstract 335: A genetic mouse model of head-and-neck squamous cell carcinoma using targeted deletion of E-cadherin and TGFβ receptor II.
Authors: Andl Thomas , Le Bras Gregoire F. , Richards Nicole F. , Allison Gillian L. , Washington M. Kay , Lee R. Katie , Andl Claudia D.
Published: August 2014

We previously observed that 70% of esophageal tumors demonstrated coordinated loss of E-cadherin and TGFβRII. When grown in three-dimensional organotypic reconstruct cultures, cells lacking E-cadherin and TGFβRII demonstrate fibroblast-dependent invasion into the underlying matrix. Therefore, we hypothesized that coordinated loss of CDH1 (E-cadherin) and TGFBR2 (TGFβRII) will induce tumorigenesis in vivo. We developed a mouse model targeting CDH1 and TGFBR2 loss in the oral-esophageal epithelium using the Epstein-Barr virus L2 promoter, ED-L2. For spatio-temporal control of CDH1- and TGFBR2 gene expression, we also generated an inducible mouse model using Cre-ERT(tam) under the control of the keratin 14 promoter. Few mouse models focusing on genetic alterations of oral and head-and-neck cancer exist. We show that the loss of E-cadherin and TGFβ receptor II without carcinogen treatment is sufficient to induce invasive HNSCC and forestomach tumors. Double knock-out animals succumb to the disease between 1 and 1.5 years of age and show invasive tumors in the oral cavity and tongue, as well as the forestomach. Advanced tumors metastasize to the lung. The tumors are characterized by Ki67-positive and p63-positive staining and show disruption of adherens junctions with loss of β-catenin and mislocalisation of p120 to the cytoplasm. Additional genetic modifications frequently described for HNSCC are the upregulation of c-myc and cyclin D1. We could show that the oral mouse tumors are positive for these markers allowing us to conclude that this animal model recapitulates HNSCC at the pathologic and molecular levels. Furthermore, the forestomach is an extension of the squamous epithelium of the esophagus in the mouse. Known genetic alterations causing esophageal squamous cancer are amplification of EGFR, cyclin D1 and mutations in p53 in addition to the loss of E-cadherin and TGFβRII. The analysis of the forestomach tumors recapitulates these events in the mouse model as a result of CDH1 and TGFBR2 loss. This tumor model will provide us with a unique tool for testing therapeutic approaches.

Citation Format: Thomas Andl, Gregoire F. Le Bras, Nicole F. Richards, Gillian L. Allison, M. Kay Washington, R. Katie Lee, Claudia D. Andl. A genetic mouse model of head-and-neck squamous cell carcinoma using targeted deletion of E-cadherin and TGFβ receptor II. [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 335. doi:10.1158/1538-7445.AM2013-335
Abstract 3367: Massbalance, excretion and metabolism of [14C]-Pralatrexate (PDX) incancer patients in a phase I trial.
Authors: Odore Elodie , Rezaï Keyvan , Madar Olivier , Weill Sophie , Awada Ahmad , Pronk Bert , Cvitkovic Esteban , Lokiec Francois
Published: August 2014

Background: PDX is a novel 10-deaza-aminopterin analogue of methotrex[[Unsupported Character - Codename ­]]ate consisting of a mixture of R- and S-diastereomeric folate derivatives. PDX inhibits folate metabolism by binding to and inhibiting the enzyme dihydrofolate reductase (DHFR). PDX is the first drug approved by the US Food and Drug Admin[[Unsupported Character - Codename ­]]istration (FDA) specifically for the treatment of patients with relapsed or refractory peripheral T-cell lymphoma. Mass balance studies in animals and humans with radiolabeled compounds represent a standard part of the development process for new drugs. Isotopically labeled analogs of a drug or its metabolites play an important role in understanding the absorption, distribution, metabolism and excretion profiles of a compound. The objectives of this study were to determine the pharmacokinetics (PK), metabolism and routes of excretion of [14C]-PDX and to characterize its metabolites in human plasma and urine.

Methods: Four patients were administered intravenously with a mixture of 50 μCi of [14C]-PDX (0.5 mg) and 224.5 mg of non radioactive PDX over a 3-5 minute infusion. Serial blood and plasma samples were drawn at: 0 (before PDX infusion), and up to 144 h after the start of infusion. Urine and fecal samples were collected for up to 168 h after the start of infusion. Expired air samples were collected up to 24 hours after administration. The radioactivity measurement in biological fluids was performed using liquid scintillation counting.

Results: Mass balance was achieved with a mean recovery radioactivity in excreta= 82.9 % ± 20.0 % for the four patients. The mean recovery of radioactivity in urine, feces, and expired air were 34.1%, 38.7% and 10.1%, respectively, indicating renal and fecal excretions were the major route of elimination of [14C]-PDX. A three-compartment open model adequately described [14C]-PDX time radioactivity-concentration courses. The AUC0-∞, and clearance values for total radioactivity in plasma were 18.1 nCi.h/mL, and 3.95 L/h, respectively. Metabolite profiling will be performed using HPLC with beta radiation detector.

Conclusions:

This study quantified the mass balance of PDX and defined its substantial excretion in both urine and feces after IV administration. The PK of [14C]-PDX was best described by a three compartment open model. The metabolite profiling of PDX is ongoing.

Citation Format: Elodie Odore, Keyvan Rezaï, Olivier Madar, Sophie Weill, Ahmad Awada, Bert Pronk, Esteban Cvitkovic, Francois Lokiec. Massbalance, excretion and metabolism of [14C]-Pralatrexate (PDX) incancer patients in a phase I trial. [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 3367. doi:10.1158/1538-7445.AM2013-3367
Abstract 3386: Mechanisms of resistance to enzalutamide in LNCaP models.
Authors: Loriot Yohann , Wyatt Alexander , Al Nakouzi Nader , Beraldi Eliana , Toren Paul , Nip Ka Mun , Gleave Martin , Zoubeidi Amina
Published: August 2014

Background

MDV3100 is a potent androgen receptor (AR) antagonist with activity in castration resistant prostate cancer (CRPC); however, progression to MDV3100-resistant (MDV-R) CRPC frequently occurs with rising serum PSA levels suggesting reactivation of AR pathway. We sought to identify AR-based mechanisms of resistance.

Methods

We treated human CRPC LNCAP-derived xenografts with MDV3100 and assessed androgen receptor (AR) levels and localization as well as genomic alterations versus controls. We also generated by selection MDV3100-R LNCaP-derived sub-lines to study mechanisms of resistance to MDV3100.

Results

Serum and tumoral PSA levels were consistently increased in resistant xenogratfs as compared with sensitive xenografts along with AR nuclear localization. MDV3100 did not induce any new mutation in the DNA binding domain and any AR DNA gene amplification. AR full-length (ARfl) and AR-V7 splicing variant were both highly expressed in RNA sequencing compared to CRPC LNCaP xenografts. However, it was no enrichment of AR-V7 versus ARfl in LNCaP MDV-R xenografts. Interestingly, AR was found in the nucleus and constitutively binding to DNA on androgen response element on PSA enhancer as measured by Chromatin immunoprecipitation in MDV-R cell lines compared to CRPC cell lines. These data were translated by increase of AR responsive genes at both mRNA and protein levels.

Conclusions

MDV-R LNCaP cells exhibited AR overexpression without any significant genomic alterations. While MDV3100 induces both ARfl and AR-V7 levels, no enrichment of AR-V7 was detected. Immunofluorescence, CHIP and qPCR studies suggest that ARfl overexpression remains a significant mechanism of resistance to MDV3100 similar to resistance from chronic androgen depletion.

Citation Format: Yohann Loriot, Alexander Wyatt, Nader Al Nakouzi, Eliana Beraldi, Paul Toren, Ka Mun Nip, Martin Gleave, Amina Zoubeidi. Mechanisms of resistance to enzalutamide in LNCaP models. [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 3386. doi:10.1158/1538-7445.AM2013-3386

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 340: Squamous cell carcinoma in the mouse: A novel, rapidly growing, syngeneic orthotopic lung tumor model .
Authors: Giladi Moshe , Schneiderman Rosa S. , Porat Yaara , Munster Mijal , Itzhaki Aviran , Mordechovich Daniel , Cahal Shay , Kirson Eilon D. , Palti Yoram
Published: August 2014

Lung cancer is the leading cause of cancer death and accounts for 28% of all cancer death in the United States. About 80% of lung cancers are non-small cell lung carcinoma (NSCLC) of which over 20% are Squamous Cells Carcinoma (SCC). The currently available therapies, for NSCLC, are less effective against squamous cells carcinoma. While there are several established in-vivo models for lung adenoma and adenocarcinomas, there are no rapidly growing, syngeneic, orthotopic tumor models for lung SCC. The goal of the present study was to develop such a model in order to facilitate research of new treatments against SCC.

Murine SCC (KLN205) cells were mixed with matrigel and injected directly into the lungs of C57BL/6 and DBA-2 mice using the same methodology applied during the induction of Lewis Lung Carcinoma orthotopic tumors. Injection of KLN205 resulted in development of highly variable tumors with an average size of 24mm3 in DBA-2 mice after 3 weeks from inoculation. No sustainable tumors were seen in C57BL/6 mice. Cells derived from DBA/2 lung tumors (referred to as KLN205-T1) were subcultured for over 10 generations and their cytological characteristics were determined. A second round of orthotopic tumor injections using KLN205-T1 cells resulted in the formation of reproducible, solitary and localized tumors in the lungs of over 85% of the DBA/2 mice but not in C57BL/6 mice. The tumors increased in average size from 20 to 40 to 80 mm3 between days 10, 13 and 16. Comparable results were obtained while imaging tumor progression using MRI. Metastases were observed only in thoracic lymph nodes which were probably a result of cells escaping the lung during tumor injection. Immunostaining of KLN205-T1 tumors with p63 and cytokeratin 5/6 demonstrated similar characteristics as described for human SCC.

Cisplatin and paclitaxel were tested in this KLN205-T1 tumor model demonstrating its potential use for testing treatment options for SCC lung cancer. The effect of Tumor Treating Fields (TTFields) therapy with and without paclitaxel or cisplatin is currently being tested in this novel tumor model.

Citation Format: Moshe Giladi, Rosa S. Schneiderman, Yaara Porat, Mijal Munster, Aviran Itzhaki, Daniel Mordechovich, Shay Cahal, Eilon D. Kirson, Yoram Palti. Squamous cell carcinoma in the mouse: A novel, rapidly growing, syngeneic orthotopic lung tumor 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 340. doi:10.1158/1538-7445.AM2013-340
Abstract 3417: Increased anticancer effect of combining Wee1 (MK1775) and Chk1 (AZD7762) inhibitors in malignant melanoma cell lines.
Authors: Magnussen Gry I. , Florenes Vivi A. , Engesæter Birgit , Emilsen Elisabeth
Published: August 2014

Malignant melanoma is one of the most increasing forms of cancer worldwide, in addition to being notoriously resistant to therapy. Despite recent advances, there are no curative treatments once the cancer has spread (stage IV); there is thus a precarious need to find new and better treatment alternatives. Unlike normal cells, the majority of cancer cells have a defective G1/S DNA-damage checkpoint and are therefore more dependent on the G2/M checkpoint when DNA damages arise. Central in regulating the latter checkpoint is Wee1, a kinase which may stop the cell cycle by adding a negative phosphorylation on CDK1. Wee1 is regulated by Chk1 which upon DNA damage can also inhibit the complimentary counterpart of Wee1; CDC25. In recent studies, Wee1 and Chk1 have additionally been indicated as key players in regulating DNA replication in S-phase. We have previously reported that increased Wee1 protein expression was associated with malignancy in melanoma tumour samples, and that siWee1 transfection led to reduced viability in melanoma cell lines. In the present study we have combined inhibitors of Wee1 (MK1775) and Chk1 (AZD7762) in a panel consisting of both metastatic melanoma- and normal cells, including a Plexxikon resistant melanoma cell line (MelJR post3.3). We found that the combination synergistically reduced the amount of viable cells in the melanoma cell lines (∼50-80% reduction), whilst only having very moderate effect (∼10% reduction) in the normal cells. Interestingly, the reduction in number of viable cells was found in both p53WT and p53Mut cell lines, indicating that the p53 status do not predict the response to such treatment. Furthermore, combinational treatment with the inhibitors caused increased cleavage of PARP and caspases, indicative of apoptosis, as compared to MK1775 or AZD7762 treatment alone. The combination also caused a marked increase in DNA double-strand breaks, as demonstrated by augmented levels of γ-H2AX (predominantly found in S-phase cells). In line with this, preliminary results from melanoma cells grown as spheroids, revealed the same tendency, with approximately 50% reduction in spheroid volume when combining the inhibitors. In vivo experiments using melanoma cells grown in SCID mice are currently ongoing. In conclusion, our results indicate a potential new role for inhibitors of Wee1 and Chk1 in treatment of malignant melanoma.

Citation Format: Gry I. Magnussen, Vivi A. Florenes, Birgit Engesæter, Elisabeth Emilsen. Increased anticancer effect of combining Wee1 (MK1775) and Chk1 (AZD7762) inhibitors in malignant melanoma cell lines. [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 3417. doi:10.1158/1538-7445.AM2013-3417
Abstract 343: Transgenic C57/BL6 x Balb/c F1 mice expressing mitochondrial targeted catalase have less spontaneous lung cancer.
Authors: Pettan-Brewer Christina , Morton John , Treuting Piper , Ladiges Warren
Published: August 2014

Human lung cancer is a major cause of morbidity and among the most frequent and deadly malignancies throughout the world. Discrepancies in classification and characterization approaches have resulted in confusions and difficulties in the comparative pathology of mouse and human lung tumors. Currently, there are very few mouse models of spontaneous lung cancer that are available to validate human lung carcinogenesis. Male C57/BL6 x Balb/c F1 (CB6 F1) mice have a high incidence of pulmonary adenocarcinoma that increases with increasing age. For example, 28 month old males have a 52 per cent incidence, while 32 month old males have close to 80 per cent incidence. In order to determine if these tumors were driven by mitochondrial reactive oxygen species (ROS), we crossed wild type Balb/c females with transgenic males on the C57/BL6 background expressing human catalase targeted to mitochondria (mCAT). The offspring were followed through the end of their lifespan when tissues were collected for histological evaluation. Of 65 males examined, we found that those expressing mCAT had a 29 per cent incidence of lung tumors, while wild type littermates had an incidence of 71 per cent (p ≤ 0.01). The presence of mCAT had a similar effect on the incidence of lung tumors in females although the overall incidence was less than in males. Histologically, lung tumors in wild type mice had higher grade malignancy than lung tumors in mice expressing mCAT suggesting that ROS generated by mitochondria might be playing a role in driving this type of cancer in mice. We conclude that spontaneous lung adenocarcinoma in old CB6 F1 mice could be a useful model to study the anti-tumor effect of mitochondrial-targeted anti-oxidants for intervention of human lung cancer.

Citation Format: Christina Pettan-Brewer, John Morton, Piper Treuting, Warren Ladiges. Transgenic C57/BL6 x Balb/c F1 mice expressing mitochondrial targeted catalase have less spontaneous lung 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 343. doi:10.1158/1538-7445.AM2013-343

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3443: Novel small molecule CRM1 inhibitors induce nuclear accumulation of p53, phosphorylated ERK and apoptosis in human melanoma cells.
Authors: Yang Jennifer , Bill Matthew A. , Young Gregory S. , Landesman Yosef , Shacham Sharon , Kauffman Michael , Senapedis William , Kashyap Trinayan , Saint-Martin Jean-Richard , Lesinski Gregory
Published: August 2014

Inhibition of nuclear export can promote re-activation of tumor suppressor proteins (TSPs) by restoring them to the nucleus. CRM1 (chromosome region maintenance 1, XPO1) is the exclusive exporter of many TSPs. We hypothesized that CRM1 inhibition can be used as a therapeutic target in melanoma. The growth inhibitory and pro-apoptotic effects of KPT-185, KPT-276 and KPT-330, potent, small molecule, selective inhibitors of nuclear export (SINEs) were evaluated in a panel of human metastatic melanoma cell lines using an MTS assay and Annexin V/PI staining, respectively. The weak CRM1 inhibitor, KPT-185 trans-isomer, and DMSO vehicle were controls in all assays. CRM1 protein was expressed in melanoma cell lines regardless of molecular profile (BRAF, NRAS, TP53) as determined by immunoblot. SINEs inhibited cell growth in a concentration-dependent manner and induced apoptosis at nanomolar concentrations (range=139.7nM - 2.41μM). Both BRAF wild type (WT) and BRAF V600E mutant lines were sensitive to apoptosis by SINE, but BRAF V600E lines had an 8.8-fold lower IC50s as compared to BRAF (WT) lines (p=0.018). The cytostatic and pro-apoptotic effects of CRM1 inhibition were associated with nuclear accumulation of p53, and p21 induction in A375 and CHL-1 melanoma cell lines with functional p53 at time points prior to apoptosis, while pERK accumulated in melanoma cells regardless of p53 status. In pharmacokinetic studies in mice, KPT-276 and KPT-330 showed >50% oral bioavailability with Cmax >5μM. Mice bearing either A375 (BRAF V600E) or CHL-1 (BRAF WT) melanoma xenografts were given KPT-276 or KPT-330 SINE by oral gavage. A375 xenografts (p<0.0001 for both drugs) and CHL-1 xenografts (p=0.0087 and 0.0002 respectively) showed significant growth inhibition as compared to vehicle-treated controls with good tolerability. KPT-330 is currently in Phase 1 clinical trials in patients with advanced cancers including melanoma as the first orally bioavailable, SINE CRM1 antagonist.

Citation Format: Jennifer Yang, Matthew A. Bill, Gregory S. Young, Yosef Landesman, Sharon Shacham, Michael Kauffman, William Senapedis, Trinayan Kashyap, Jean-Richard Saint-Martin, Gregory Lesinski. Novel small molecule CRM1 inhibitors induce nuclear accumulation of p53, phosphorylated ERK and apoptosis in human melanoma 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 3443. doi:10.1158/1538-7445.AM2013-3443
Abstract 3456: Macrophages as independent prognostic factors in small T1 breast cancers.
Authors: Carrio Roberto , Koru-Sengul Tulay , Miao Feng , Gluck Stefan , Lopez Omar , Selman Yamil , Alvarez Consuelo , Milikowski Clara , Gomez Carmen , Jorda Merce , Nadji Mehrad , Torroella-Kouri Marta
Published: August 2014

Breast cancer is the second leading cause of death by cancer in women in the United States. The occurrence of high numbers of macrophages in the tumor stroma has been associated with tumor progression and poor prognosis in breast and other solid malignances. However, macrophage numbers in tumors have not been validated as a prognostic factor in clinical practice. The present analysis was designed as a pilot study aimed at determining whether the presence of CD68+ macrophages is an independent prognostic factor in small T1 ER+ breast cancers across three different ethnic groups, i.e. African American, Latinas and Caucasian women. A retrospective pilot analysis of 30 T1 breast cancers cases encompassing these three ethnic groups was carried out. African

American and Latina women present with less incidence but more aggressive breast cancer disease and therefore proportionally higher death rates. Using immunohistochemistry, we sought to find out whether there was any association between the presence and density of CD68+ macrophages and standard prognostic markers with overall survival in these groups. Our data showed that overall survival did not differ significantly for the occurrence or density of CD68+ macrophages in T1 ER+ tumors. There were also no significant differences in overall survival for the occurrence of CD68+ macrophages across ethnicities, although macrophage numbers were significantly higher in tumors from African American and Latinas than in Caucasian patients. Interestingly but not surprisingly, the absence of PR associated very strongly with decreased overall survival. This pilot project shows that CD68+ macrophages are not pivotal in determining tumor prognosis in early T1 breast cancers. New studies are presently being conducted to assess the value of different macrophage markers and macrophage activation profiles as prognostic factors in breast cancers of different clinical stages, using a larger number of patients among these three different ethnicities.

Citation Format: Roberto Carrio, Tulay Koru-Sengul, Feng Miao, Stefan Gluck, Omar Lopez, Yamil Selman, Consuelo Alvarez, Clara Milikowski, Carmen Gomez, Merce Jorda, Mehrad Nadji, Marta Torroella-Kouri. Macrophages as independent prognostic factors in small T1 breast cancers. [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 3456. doi:10.1158/1538-7445.AM2013-3456
Abstract 3484: CTC enumeration and molecular characterization in a Phase 1 study of IGF-1 and IGF-2 inhibition by MEDI-573.
Authors: Chen Xiaoru , Shi Xiaoqing (Sarah) , Bao Haifeng , LaVallee Theresa M. , Mendel Dirk B. , Burke Patricia A.
Published: August 2014

MEDI-573 selectively binds to human insulin-like growth factor (IGF)-1 and IGF-2 and inhibits ligand-mediated signal transduction via the insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor A isoform (IR-A) pathways, without perturbing glucose homeostasis. As IGF-1, IGF-2, and IGF-1R are overexpressed in bladder cancer, a Phase 1 expansion study including 20 bladder cancer subjects along with 6 additional subjects with other types of cancers from the dose escalation study were included for circulating tumor cell (CTC) analysis as part of a larger 42-subject study (MI-CP184) evaluating PK, safety, and biomarkers. Our objective for the results reported here was to evaluate CTC numbers as a prognostic indicator and as a biomarker of response to MEDI-573 therapy, and to evaluate the expression of IGF-1R on CTC and any correlation with treatment. Subjects in the expansion study were treated with MEDI-573 at 5 mg/Kg weekly (QW) (n=10), 15 mg/Kg QW (n=10), whereas subjects evaluated for CTC in the dose escalation study were treated at 30 or 45 mg/Kg every 3 weeks (Q3W) (n=3 each). CTC collected at screening and pre-dosing each cycle were enumerated and evaluated for IGF-1R expression by CellSearch®. 92% of subjects had at least one CTC at any time during the study and 62.5% had at least one CTC at screening. As has been demonstrated with other tumor types, subjects with ≤3 CTC at screening stayed on study longer than subjects with >3 CTC (2.3 vs. 1.2 months, P=0.0125 [no censoring]). Mean overall survival (OS) was also longer for subjects with ≤3 CTC compared to subjects with >3 CTC (6.37 vs. 2.83 months (P=0.0218, t test), although Kaplan-Myer curves were not different (P=0.0854)). Subjects with decreased numbers of CTC or no change in CTC with treatment stayed on study longer (not significant, P=0.0512, t test) than subjects with increased CTC with treatment (2.95 vs. 1.70 months). 73.3% (11/15) of subjects with positive CTC at screening had at least 1 CTC positive for IGF-1R. In conclusion, our results provide evidence for the utility of CTC as prognostic indicators in a Phase 1 study.

Citation Format: Xiaoru Chen, Xiaoqing (Sarah) Shi, Haifeng Bao, Theresa M. LaVallee, Dirk B. Mendel, Patricia A. Burke. CTC enumeration and molecular characterization in a Phase 1 study of IGF-1 and IGF-2 inhibition by MEDI-573. [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 3484. doi:10.1158/1538-7445.AM2013-3484
Abstract 3516: AZD1208 PIM kinase inhibitor - Preliminary evidence of target pathway inhibition in Phase I clinical trials of AML.
Authors: McEachern Kristen A. , Cao Yichen , DuPont Rachel , Pablo Lourdes , McCoon Patricia , Cortes Jorge E. , DeAngelo Daniel J. , Minden Mark D. , Hewes Becker , Brown Jeffrey L. , Barrett Carl
Published: August 2014

PIM kinases have been shown to play a key role as downstream effectors of growth factor signalling pathways including Flt3 and the Jak-STAT signalling pathways in AML, NHL and other solid tumors. AZD1208 is a novel, orally bioavailable, highly selective PIM kinase inhibitor with single nanomolar potency against all three PIM kinases and is currently undergoing Phase I testing and dose escalation studies in AML. Here we describe a multiplexed biomarker strategy measuring pBAD, p4EBP1 and p-p70S6K as downstream pharmacodynamic biomarkers for PIM kinase inhibition in clinical trials. Patient bone marrow aspirates and peripheral blood samples were collected pre- and post-AZD1208 treatment in AML patients during the Phase I dose escalation and expansions. Primary patient samples were analyzed for quantitative changes in pBAD, p4EBP1 by NanoPro and MesoScale assay platforms as well as a qualitative evaluation of p-p70S6K and other exploratory endpoints. Preclinical PK-PD modeling data with AZD1208 had suggested that greater than a 50% decrease in the levels of one of these phosphorlyated substrates would be indicative of efficacy and PIM pathway inhibition. Following a single dose of AZD1208 at 120mg, the first dose level, approximately 60-70% inhibition of pBAD, S112 was seen in the bone marrow and peripheral blasts. Taken together, the data presented here provide evidence for single agent AZD1208 activity in AML patients based on quantitative reduction in these biomarkers. Correlations of these biomarker endpoints with Phase I pharmacokinetic data underscore the therapeutic potential of Pim kinase inhibition by AZD1208 for the treatment of AML, and strongly support further investigation of this agent in other indications where PIM signaling may play a role in tumorigenesis and survival.

Citation Format: Kristen A. McEachern, Yichen Cao, Rachel DuPont, Lourdes Pablo, Patricia McCoon, Jorge E. Cortes, Daniel J. DeAngelo, Mark D. Minden, Becker Hewes, Jeffrey L. Brown, Carl Barrett. AZD1208 PIM kinase inhibitor - Preliminary evidence of target pathway inhibition in Phase I clinical trials of AML. [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 3516. doi:10.1158/1538-7445.AM2013-3516
Abstract 3541: Significance of osteopontin in the sensitivity of malignant pleural mesothelioma cells to pemetrexed.
Authors: Seike Masahiro , Takeuchi Susumu , Soeno Chie , Noro Rintaro , Minegishi Yuji , Gemma Akihiko
Published: August 2014

Introduction: Malignant pleural mesothelioma (MPM) is one of the fatal cancers associated with asbestos exposure.Pemetrexed (PEM) is currently recommended as one of the antitumor drugs consisted of standard therapies for MPM.However, the mechanism of the sensitivity of MPM to PEM remains unclear. Methods: We analyzed the antitumor effects of PEM in six MPM cell lines by MTS assay. To identify genes associated with drug sensitivity,we conducted gene expression profiling on the same set of cell lines using Affymetrix HG-U133A GeneChips. Pathway analysis was performed to provide a viewpoint of the biological function of the genes. Results: Three cell lines were sensitive to PEM. Eighteen transcripts and fourteen genes identified by Gene-chips were significantly correlated with sensitivity to PEM. Pathway analysis revealed that SPP1/OPN was an important target in PEM sensitivity. Overexpression of SPP1 was observed in the sensitive cells by Western blotting. Introduction of SPP1 by lentiviral vector significantly enhanced the invasion activities of MPM cells. Expression of SPP1/OPN and AKT phosphorylation were significantly decreased after PEM treatment of the PEM-sensitive cells. PEM treatment with SPP1/OPN knockdown inhibited the PEM-induced cell growth inhibitory effect in PEM-sensitive cells. Conclusions: Our results suggest that SPP1 may be used as a single predictive biomarker of the effectiveness of PEM treatment in MPM. Patient selection based on SPP1/OPN expression may be useful for future clinical development of PEM therapy for MPM.

Citation Format: Masahiro Seike, Susumu Takeuchi, Chie Soeno, Rintaro Noro, Yuji Minegishi, Akihiko Gemma. Significance of osteopontin in the sensitivity of malignant pleural mesothelioma cells to pemetrexed. [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 3541. doi:10.1158/1538-7445.AM2013-3541