bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C50: AST-6, a novel irreversible inhibitor of the epidermal growth factor receptor 1 and 2, inhibits tumor growth both in vitro and in vivo
Authors: Xie Hua , Lin Lipin , Tong Linjiang , Jiang Yong , Wang Yi , Zhang Xiaowei , Geng Meiyu , Guo Jianhui , Ding Jian
Published: June 2012

Despite the initial response to the reversible, ATP-competitive quinazoline inhibitors that target ErbB-family, such a subset of cancer patients almost invariably develop resistance. Recent compelling evidence demonstrated that irreversible ErbB inhibitors were significantly active to override this resistance. Several irreversible inhibitors are now under preclinical or clinical studies. Here, we found that AST-6, a rationally designed novel anilinoquinazoline compound, inhibited enzymatic activities of wild type EGFR and ErbB2 as well as EGFR L858R/T790M resistant mutant both in cell-free and cell-based levels. Importantly, AST-6 functions as an irreversible inhibitor, most likely by covalent modification of Cys 797 and Cys 805 in the catalytic domains of EGFR and ErbB2, respectively. Further studies showed that AST6 inactivated their downstream pathways and thereby inhibited the proliferation of a panel of cancer cell lines. Detailed insights manifested that ErbB2 expression experienced with a consistently higher sensitivity to AST-6 across various cell lines. And in parallel, ErbB2, but not EGFR, silencing in turn decrease the sensitivity of SK-OV-3 cells to AST-6. As a consequence, AST-6 caused a potent tumor growth inhibition in ErbB2-overexpressed SK-OV-3 human ovarian carcinoma and Calu-3 lung adenocarcinoma xenograft models, but weak suppression in EGFR-overexpressed tumor xenografts. The above findings, combined with a significant restriction of tumor growth challenged by AST6 in FVB-2/Nneu transgenic breast cancer mouse model, define AST-6 as a selective irreversible dual ErbB2 and EGFR inhibitor with ErbB2 being more sensitive.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C50.
Abstract C53: Dose-finding designs for combination therapies involving molecularly targeted agents
Authors: Mandrekar Sumithra J. , Qin Rui , Sargent Daniel J.
Published: June 2012

Purpose: Novel therapies are challenging current drug development standards. Agents with specific biologic targets, unknown dose-efficacy curves, limited toxicity profiles, and in combination therapy require novel statistical designs. We compared the operating characteristics of three model based designs incorporating both toxicity and a measure of efficacy to identify the optimal dose region for combination therapies.

Methods: Three designs were considered: continuation ratio model with a trinary outcome, bivariate logistic model treating toxicity and efficacy as independent outcomes, and bivariate probit model allowing for dependent toxicity and efficacy outcomes. All designs utilized a model-based methodology to adaptively determine dose escalation/de-escalation, based on the data observed to date in the trial. The model parameters were estimated using Markov chain Monte Carlo approach in a Bayesian framework. The target threshold for toxicity was set at 33%; the minimum and maximum sample sizes were set at 30 and 45, respectively, for the simulated trials; and the cohort size was 3 with at least nine patients needed to be treated at the proposed combination dose level. The performance of the designs was evaluated in simulation studies under six clinically relevant scenarios.

Results: As expected, the ability to correctly identify an optimal dose region was sensitive to the changes in the toxicity and/or efficacy across the dose level combinations. Considering 50% as a benchmark for the recommendation rate for the optimal dose or region (dose levels within 10% of the maximum efficacious dose level), at least one of the three models performed well in recommending the optimal dose region except when the dose-efficacy surface was non-monotone or the dose-toxicity surface was close to the target rate of 33%. All three models worked well in not recommending any dose combination when the starting dose level combination was too toxic. The median sample size across all scenarios ranged from 27–33.

Conclusions: Despite favorable operating characteristics, the implementation of these new designs in practice is challenging for a variety of reasons. Adoption of novel designs require a multi-pronged approach that provides a) clinically obvious advantages, b) practical design framework with a transparent decision making process, c) timely access to both toxicity and preliminary efficacy data, and d) portable user-friendly software, or very attentive statisticians.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C53.
Abstract C56: NCCTG phase I trial of everolimus (RAD001) and temozolomide (TMZ) in combination with radiation therapy (RT) in newly diagnosed glioblastoma multiforme (GBM) patients
Authors: Sarkaria Jann N. , Galanis Evanthia , Peller Patrick , Brown Paul , Wu Wenting , Felten Sara , Uhm Joon , Giannini Caterina , Jaeckle Kurt , Buckner Jan
Published: June 2012

Background: The mammalian target of rapamycin (mTOR) functions within the PI3K/Akt signaling pathway as a critical modulator of cell survival. We previously demonstrated significant synergy of the mTOR inhibitor sirolimus with RT in glioma xenografts. This Phase I study evaluated the safety and tolerability of combined therapy with the mTOR inhibitor RAD001, RT and TMZ.

Methods: All patients received weekly oral RAD001, starting 1 week prior to RT/TMZ and continuing until progression, with a break from therapy between RT/TMZ and adjuvant TMZ. RAD001 was dose escalated in cohorts of 6 patients. In addition to RAD001, all pts received standard treatment with RT (60 Gy)/TMZ (75 mg/m2 daily) and adjuvant TMZ (200 mg/m2 daily × 5 every 28 days). DLTs were defined during therapy with RAD001 combined with TMZ/RT.

Results: A total of 18 patients were enrolled. Combined RAD001/RT/TMZ was well tolerated at all dose levels with 1 of 6 patients with a DLT at each dose level. DLTs were Grade 3 fatigue (RAD001 25 mg/week), Grade 4 hematologic toxicity (RAD001 50 mg/week), and Grade 4 liver function elevation (RAD001 70 mg/week). With adverse event information available for all 18 patients, no grade 5 events were seen. Nine grade 4 events (thrombosis, fatigue, anemia, neutropenia, thrombocytopenia, leukopenia, SGPT (ALT), SGOT (AST), bilirubin elevation) were seen in 3 patients. On the basis of these results, the recommended Phase II dose currently being tested is RAD001 70 mg/week in combination with standard RT/TMZ and adjuvant TMZ. FDG PET scans were also obtained prior to the first dose of RAD001 and within 24 hours of the second dose of RAD001, prior to starting RT or TMZ, and changes in maximum standard uptake values (SUV) were compared between scans for each patient. Two patients at 25 mg RAD001/week had a 21% and 24% change in SUV, and 2 patients receiving 70 mg/week had a change in SUV uptake of −33% and −16%. Additional follow-up will be required to evaluate whether these SUV changes are associated with outcome.

Conclusions: RAD001 in combination with RT/TMZ and adjuvant TMZ was well tolerated, and the imaging results support continued evaluation of PET imaging as an early predictor of response to therapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C56.
Abstract C6: Phase 1 study of conatumumab (AMG 655), a proapoptotic death receptor-5 agonist antibody, in Japanese patients with advanced solid tumors
Authors: Murakami H. , Yamamoto N. , Boku N. , Onozawa Y. , Doi T. , Ohtsu A. , Fuse N. , Yoshino T. , Saito H. , Sasaki T. , Hsu M. , Gorski K. , Friberg G.
Published: June 2012

Background: Conatumumab is an investigational, fully human monoclonal agonist antibody that binds human death receptor-5 (DR5, TR-2), activates caspases, and induces apoptosis in sensitive tumor cells. The primary objectives of this phase 1 study were to assess the safety, tolerability, and pharmacokinetics (PK) of conatumumab in Japanese patients (pts) with advanced solid tumors.

Method: This is an open-label ascending dose study with a starting dose level of 3 mg/kg. Subsequent doses of 10 mg/kg and 20 mg/kg were planned. Six pts were enrolled into 1 of 3 dose cohorts (3, 10, or 20 mg/kg) of conatumumab administered intravenously once every 2 weeks as a single-agent. No conatumumab was administered on day 43 to allow assessment of terminal PK parameters. Conatumumab receptor occupancy on monocytes was assessed during the first treatment cycle. Pts remained on study until tumor progression or unacceptable toxicities occurred.

Results: Eighteen pts (6 in each dose cohorts) received at least 1 dose of conatumumab; 9 pts were men; median age was 58 years. Tumor types included: colorectal (5), non-small cell lung (4), gastric (2), soft tissue sarcoma (2) and one each of pancreatic cancer, thymoma, mesothelioma, rectal carcinoid and parotid cancer. The grade 3 adverse events included blood creatine phosphokinase increase, pain and increased lipase. One treatment emergent serious adverse event of pain was reported in cohort 2. This serious adverse event was not considered by the investigator to be related to conatumumab. There were no DLTs observed as defined in the protocol. No deaths occurred during treatment or the safety follow-up period. No subjects had an adverse event leading to conatumumab discontinuation. Conatumumab demonstrated dose-linear kinetics with a half-life of 12 to16 days. Pharmacokinetic profiles were similar to those in the Caucasian population. Tumor response data were available for all patients. A best response of stable disease (defined as a lack of progression at the first assessment at 8-weeks) was reported in 9 patients and progressive disease in 9 patients. Monocytes were found to express DR5, and showed a high degree of conatumumab receptor occupancy after treatment at all dose levels.

Conclusions: Conatumumab administered up to 20 mg/kg once every 2 weeks did not result in dose limiting toxicities in Japanese patients with advanced solid tumors. Adverse events and pharmacokinetics were similar to those in the first in human study conducted in the United States.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C6.
Abstract C61: The PI3K pathway inhibition in prostate cancer cells is enhanced by dual targeting with the mTOR inhibitor RAD001 and a mTOR/PI3K inhibitor leading to decreased cell proliferation and increased autophagy
Authors: Balakumaran Bala S. , Herbert James T. , Glover Wayne , Foye Adam , Pierobon Mariaelena , Petricoin Emanual , George Dan , Febbo Phillip G.
Published: June 2012

Introduction: Metastatic prostate cancer patients often become resistant to standard treatments such as androgen ablation therapy and docetaxel treatment and novel treatments are needed. Loss of PTEN and subsequent activation of the PI3K pathway is common in patients with advanced prostate cancer. However, use of mTOR inhibitors such as rapamycin analogues as single agents has had limited clinical impact and the identification of a potent therapeutic strategy for PI3K inhibition remains a critical question.

Experimental procedures: We have used gene expression data generated from metastatic tumors collected from patients with advanced prostate cancer both before and after treatment with RAD001 along with in vitro experiments in established prostate cancer cell lines to determine the impact of TORC1 inhibition, PI3K kinase inhibition, and dual inhibition on PI3K pathway signaling and cancer cell kinetics.

Summary: Treatment with RAD001 decreased tumor levels of pS6 but failed to result in a single clinical response. Gene set enrichment analysis of metastatic castration-resistant prostate cancer biopsies prior to and following treatment with RAD001 was associated with increased PI3K activity following treatment. Analysis of the activity of the downstream components of the PI3K/AKT pathway following RAD001 treatment showed reduced pS6 levels, had minimal impact on p4EBP1 levels, and increased phosphorylation of AKT1. Cytotoxic synergy is observed when RAD001 is combined with the PI3K inhibitor, LY294002 and dual PI3K/mTOR kinase inhibitors. These combinations result in more profound reduction of TORC1 activity resulting in decreased cell proliferation and increased autophagy but a continued persistence of pAKT. Reverse phase proteomics supports increased AKT activity in an androgen-receptor positive prostate cancer cell line treated with the combination as multiple AKT targets experience increased phosphorylation.

Conclusion: TORC1 inhibition results in an upregulation of PI3K activity and therapeutic combinations that inhibit both TORC1 and PI3K demonstrate cytotoxic synergy. The signaling manifestations of these combinations are a profound inhibition of TORC1 activity together with persistent phosphorylation and activity of AKT.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C61.
Abstract C64: Treatment with the Pim protein kinase inhibitor SMI-4a enhances AMPK phosphorylation, decreases Raptor levels, and blocks mTORC1 activity
Authors: Kraft Andrew S. , Beharry Zanna , Lin Ying-Wei , Mahajan Sandeep , Zemskova Marina , Xia Zuping , Smith Charles
Published: June 2012

We have identified a class of small molecule inhibitors of the Pim protein kinases, benzylidene thiazolidine-2-4 diones (J. Med. Chem. (2009) 52:74) with the most potent members having IC50s of 13 nM for Pim-1 and 2.3 µM for Pim-2. Compounds in this chemotype demonstrated selectivity of more than 2500-fold and 400-fold for Pim-1 or Pim-2 respectively while other congeners had equivalent potency towards both isozymes. In vivo, these molecules inhibited Pim kinase autophosphorylation and in a murine model inhibited the growth of subcutaneously implanted murine adenocarcinoma JC cells. One of the members of this chemotype, SMI-4a, has been shown to block the phosphorylation of the mTOR regulatory protein PRAS40 and subsequently the activity of the mTOR pathway (Mol. Cancer. Ther. (2009) 8: 1473; Cancer Biol. Ther. (2009) 8: 846). Now we show that the addition of SMI-4a to malignant cells increases the phosphorylation of AMPKα on Thr 172 in a LKB1-dependent manner, induces the phosphorylation of Raptor on Ser792, decreases the levels of Raptor protein, and inhibits mTORC1 activity. Immunoprecipitation of mTOR from SMI-4a treated cells consistently showed lower levels of bound Raptor and in vitro mTOR kinase assays from treated cells demonstrated a decreased ability to phosphorylate 4E-BP1. Knockdown of PIM-1 via siRNA in K562 leukemic cells showed increased AMPK phosphorylation and decreased Raptor protein levels, further demonstrating an important role for Pim kinase in regulating AMPK phosphorylation and Raptor levels. Additionally, mouse embryo fibroblasts (MEFs) deficient for Pim-1, Pim-2 and Pim-3 kinase (TKO MEFs) showed a significantly increased level of AMPK phosphorylation compared to wild type MEFs, which correlated with decreased mTORC1 activity and increased binding of 4E-BP1 with eIF-4E. The TKO MEFs grew significantly more slowly than wild type. The decreased mTORC1 activity correlated with an increase in the cellular level of AMP in TKO MEFs. Furthermore, the correlation between increased AMPK phosphorylation and a lower level of Raptor protein observed with SMI-4a treatment was also observed in TKO MEFs. Infection of TKO MEFs with lentiviruses expressing Pim 1 or Pim2 was able to reverse these effects, decreasing AMPK phosphorylation, and increasing Raptor protein levels. The cellular activity of mTORC1 was difficult to assess in TKO MEFs as we found substantially lower protein levels of the mTORC1 substrates 4E-BP1 and p70S6K. Akt was readily phosphorylated upon serum stimulation of TKO MEFs and mTORC2 activity was unchanged. Given the role of Pim kinase in regulating mTORC1 activity, we have combined SMI-4a and the mTOR inhibitor rapamycin inducing synergistic blockade of this pathway.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C64.
Abstract C65: Enhanced antitumor effect by the combination of a PI3K inhibitor ZSTK474 with an mTOR inhibitor rapamycin
Authors: Yoshimi Hisashi , Okamura Mutumi , Yamazaki Kanami , Yamori Takao
Published: June 2012

PI3K is a promising molecular target for the therapy of cancer. Various PI3K inhibitors are now under development. We developed a novel PI3K inhibitor ZSTK474 and demonstrated its potent antitumor efficacy. ZSTK474 is highly specific to PI3K and does not inhibit other kinases including mTOR that is a down stream component of PI3K pathway. The purpose of this study was to examine whether dual inhibition of PI3K and mTOR further improved antitumor efficacy. To assess the effect of the combination of ZSTK474 and rapamycin on prostate cancer PC-3 cells and lung cancer A549 cells in vitro, we used the isobologram method. The combination of ZSTK474 and rapamycin showed a supra-additive effect (synergism) on both PC-3 and A549.

We then evaluated the efficacy of the combination of ZSTK474 with an mTOR inhibitor rapamycin against the xenografts of PC-3 and A549. Suboptimal doses of ZSTK474 and rapamycin were determined as 100 mg/kg/day (p.o., from day 0 to day 14, except days 3 and 10) and 0.1 mg/kg/day (i.p., from day 0 to day 5 except day 3), respectively. Then, the combination of the two drugs was compared to the single drug treatments. The combination of ZSTK474 and rapamycin showed almost complete inhibition of the tumor growth whereas each single drug treatment showed partial inhibition. The synergy in the toxicity was not observed. We examined the effects of the combination on the PI3K/Akt downstream molecules in the PC-3 tumor tissues by immunohistochemical technique. The single treatment of ZSTK474 reduced the phosphorylation of Akt (Ser473), GSK3-beta and 4E-BP1, and partially reduced the phosphorylation of S6 and the expression of Cyclin D1. The single treatment of rapamycin reduced the phosphorylation of 4E-BP1, and partially reduced the phosphorylation of S6 and the expression of Cyclin D1, but did not reduce the phosphorylation of Akt (Ser473) or that of GSK3-beta. The combination of ZSTK474 and rapamycin reduced the phosphorylation of Akt, GSK3-beta, 4E-BP1 and S6, and the expression of Cyclin D1. These results confirmed the therapeutic efficacy of the combination of ZSTK474 and rapamycin against PC-3 xenografts. Therefore, the combination of PI3K inhibitor and mTOR inhibitor may be effective for cancer therapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C65.
Abstract C67: JFCR39 COMPARE system can predict molecular targets of phophatidylinositol 3-kinase inhibitors with high resolution
Authors: Kong Dexin , Dan Shingo , Yamazaki Kanami , Yamori Takao
Published: June 2012

JFCR39 COMPARE system is an anticancer drug-activity database that we established by exploiting a panel of 39 human cancer cell lines. Molecular target of an agent can be predicted by comparing its cell growth inhibition profiles (fingerprint) with those of anticancer drugs using the COMPARE program. As accumulating evidences suggest close involvement of phosphatidylinositol 3-kinase (PI3K) in various diseases, particularly in cancer, researchers in both industry and academics are in a race to develop PI3K inhibitors. Consequently, novel PI3K inhibitors such as ZSTK474, GDC-0941, NVP-BEZ235 and PI-103 have been developed in recent years. Even though all these inhibitors were reported to inhibit class I PI3K but not dozens of protein kinases, whether they have different molecular targets remained unknown. To predict such molecular target specificity, we have examined the fingerprints of these novel PI3K inhibitors and classical PI3K inhibitor LY294002 across JFCR39 panel, and analyzed the results using the COMARE system. Interestingly, we found highly similar fingerprints for ZSTK474 and GDC-0941 and cluster analysis distinguished them from the other three PI3K inhibitors, suggesting that ZSTK474 shared more in molecular targets with GDC-0941 than with any other PI3K inhibitors. To test this prediction, we determined their effects on PI3K superfamily (including classes I, II, and III PI3Ks, PI4K and PI3K-related kinases) by using several novel non-radioactive biochemical assays. As a result, ZSTK474 and GDC-0941 indicated highly similar inhibition profiles for PI3K superfamily, with class I PI3K inhibitory specificity much higher than the other three inhibitors, which supported the hypothesis derived from JFCR39 COMPARE result. In conclusion, JFCR39 system is able to provide valuable information regarding molecular target specificity, suggesting its utility in target identification with high resolution.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C67.
Abstract C75: A novel multi-targeted Aurora A and VEGFR2 kinase inhibitor, ENMD-2076, demonstrates synergistic antiproliferative and proapoptotic effects in combination with chemotherapy and trastuzumab in breast cancer cell lines
Authors: Diamond Jennifer R. , Eckhardt S Gail , Pitts Todd M. , Britt Blair C. , Kachaeva Maria I. , Varella-Garcia Marileila , Bray Mark R. , Fletcher Graham C. , Tentler John J.
Published: June 2012

Introduction: ENMD-2076 is an orally bioavailable small molecule kinase inhibitor with antiproliferative activity via Aurora A kinase inhibition, and antiangiogenic activity via VEGFR2 inhibition. ENMD-2076 is being studied in a phase I clinical trial in advanced solid tumors and has demonstrated a tolerable side-effect profile. Due to the involvement of both Aurora A and angiogenic signaling pathways in breast cancer, we explored the combination of ENMD-2076 and chemotherapy or anti-HER2 therapy against a panel of breast cancer cell lines.

Methods: Breast cancer cell lines were exposed to varying submicromolar concentrations of ENMD-2076 alone and in combination with carboplatin, docetaxel, or trastuzumab (HER2-overexpressing cell lines only). Proliferation was assessed using an SRB assay and analyzed using the Calcusyn program, whereby synergy was defined as a Combination Index of less than 1. FISH was performed to assess Aurora A gene amplification, VEGF concentrations were measured by ELISA, and apoptosis was analyzed using a caspase 3/7 assay.

Results: The combination of ENMD-2076 and carboplatin resulted in synergistic inhibition of proliferation in both HER2 + (BT-474, SKBr3, ZR-75-30) and in triple negative (MDA 231, MDA 468) breast cancer cell lines. Likewise, ENMD-2076 and trastuzumab resulted in robust synergistic growth inhibition in HER2 + breast cancer cell lines. Synergy was not observed with the ENMD-2076 and docetaxel combination. FISH analysis demonstrated that tumor cells were sensitive to ENMD-2076 regardless of levels of Aurora A amplification. An increase in apoptosis that was maximal at 72 hours was observed with single agent ENMD-2076 and the combinations with carboplatin or trastuzumab. Similarly, VEGF concentrations were decreased in response to treatment with ENMD-2076 alone or in combination with the other agents.

Discussion: These in vitro results demonstrate that ENMD-2076 acts synergistically with carboplatin to inhibit proliferation of triple negative breast cancer cell lines, and with both carboplatin and trastuzumab towards HER2+ breast cancer cell lines. This activity is associated with induction of cell death and reduction of VEGF secretion. These data are being confirmed in vivo using both trastuzumab-sensitive and trastuzumab-resistant xenograft models.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C75.
Abstract C81: Preclinical evaluation of riluzole for the treatment of mantle cell lymphoma
Authors: Medina Daniel , Zheng Laura , Rafanan Marites , Goydos James , Strair Roger
Published: June 2012

Purpose: Emerging evidence suggests a role for glutamate and its receptors in the pathogenesis of several forms of cancer. Investigators from our institution have recently shown that the metabotropic glutamate receptor 1 plays a significant role in the pathogenesis of melanoma and that inhibition of this pathway by the glutamate release inhibitor Riluzole results in suppression of cell growth. In this study we test the hypothesis that inhibition of metabotropic glutamate receptors by Riluzole in mantle cell lymphoma cells will result in a reduction of cell proliferation/survival.

Experimental Design: Mantle cell lymphoma (MCL) cell lines (MINO, JEKO and Rec-1) were treated with several concentration of the glutamate release inhibitor Riluzole. Samples were collected after 24, 48 and 72 hours of treatment and evaluated for cell viability and cell number by trypan blue exclusion, and cell proliferation and cell cycle analysis by flow cytometry. Apoptosis was evaluated by PARP cleavage and staining for Annexin V.

Results: All three MCL cell lines treated with Riluzole demonstrated a significant decrease in cell number and cell viability with an IC50=25uM. MCL cells treated with Riluzole showed a block in S and G2/M of the cell cycle followed by apoptosis. Apoptosis was confirmed by annexin V expression and by PARP cleavage.

Conclusions: Our date demonstrates that glutamate blockade with Riluzole results in the inhibition of mantle cell lymphoma proliferation, cell viability and the induction of cell cycle arrest at S and G2/M phases of the cell cycle. In addition, we show that the loss of cell viability is due to the activation of the apoptotic pathway. These preliminary findings suggest that expression of metabotropic glutamate receptors may be important in the pathogenesis and that targeting this pathway may provide a novel and effective therapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C81.
Abstract C86: FGFR inhibition potentiates the effect of conventional chemotherapeutic agents in endometrial cancer cells
Authors: Byron Sara A. , Gartside Michael G. , Pollock Pamela M.
Published: June 2012

Endometrial cancer is the most common gynecological malignancy and the fourth most common cancer in women in the United States. Most women present with early stage disease confined to the uterus and thus have a favorable prognosis. However, for the subset of women who present with advanced disease or that recur, prognosis is poor, with a median survival after recurrence of 10 months, reflecting a lack of effective treatment options. We identified activating mutations in the fibroblast growth factor receptor 2 (FGFR2) receptor tyrosine kinase in 10–15% of endometrial tumors, and hypothesized that FGFR inhibition may be a viable therapeutic option for patients with FGFR2 mutation positive tumors. Indeed, we have shown that treatment of FGFR2 mutation positive endometrial cancer cell lines with PD173074, a pan-FGFR small molecular inhibitor, induces cell death both in vitro and in vivo. We have shown this cell death is caspase-independent, raising the possibility that combination of an FGFR inhibitor with chemotherapeutic agents may exhibit synergistic activity due to their non-redundant mechanisms of action. Moreover, as FGF ligand expression has been associated with chemoresistance and poor prognosis in a number of other cancer types, including lung, breast, ovarian, prostate, and head and neck carcinomas, we hypothesized that FGFR inhibition may also sensitize wildtype FGFR2 endometrial cancer cell lines to chemotherapeutic agents. We therefore sought to evaluate whether FGFR inhibition synergizes with conventional chemotherapeutic agents in endometrial cancer. Using combination index and Bliss additivity analysis, we evaluated whether FGFR inhibition potentiated the effect of paclitaxel and doxorubicin in a panel of FGFR2 mutant and non-mutant endometrial cancer cell lines. FGFR2 mutation status did not alter sensitivity to either chemotherapeutic agent alone. The combination of PD173074 with paclitaxel or doxorubicin showed synergistic activity in all three FGFR2 mutant cell lines. In addition, though non-mutant cell lines were resistant to FGFR inhibition alone, the addition of PD173074 potentiated the effect of paclitaxel and doxorubicin in a subset of FGFR2-wildtype cell lines tested. Together these data suggest a therapeutic benefit to combining an FGFR inhibitor with standard chemotherapeutic agents, and, importantly, this may extend to patients whose tumors possess wildtype FGFR2.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C86.
Abstract C9: Utility of serological apoptosis biomarkers: Assessment in a phase II study of zibotentan (ZD4054) in patients with hormone-resistant prostate cancer
Authors: Malone Mike , Mann Helen , Growcott Jim , Butt Fouziah , Moore David
Published: June 2012

Serological apoptosis biomarkers, cytokeratin-18 (M65), cleaved cytokeratin 18 (M30) and nucleosomal DNA (nDNA) are frequently elevated above normal levels in patients with various cancers and are considered to reflect tumor burden.1 Moreover, these biomarkers appear to be dynamically modulated by cytotoxic therapy capable of inducing tumor response.2–4 We retrospectively investigated whether a non-cytotoxic agent, zibotentan, had any impact on serological levels of these apoptosis biomarkers. Zibotentan has shown survival benefit in hormone-resistant prostate cancer (HRPC) patients despite the lack of RECIST based responses.5

Serological samples were obtained from consented patients before the start and at 12 weeks after the assigned randomized treatment (placebo, zibotentan 10 or 15 mg daily oral doses) and were processed for M30, M65 and nDNA concentrations. Approximately 59% (N=184) and 42% (N=82) of the study intent to treat population (N=312 and N=195) had M30 values at baseline and at week 12, respectively. Similar numbers of M65 and nDNA values were available for analysis.

No consistent dose-related trends in mean percentage change from baseline or at 12 weeks were observed for any of the biomarkers. However, there were trends towards reductions for M30 in patients receiving zibotentan 10 mg (−11%, 95% confidence interval [CI] −24%, +6%) and for nDNA in patients receiving zibotentan 15 mg (−15%, 95% CI −44%, +30%). Applying a Cox proportional hazards regression model for overall survival (OS) and including a treatment factor and baseline covariates, stratifying the apoptosis biomarkers on the basis of ‘high’ or ‘low’ concentrations (based on median baseline values) revealed a statistically significant difference for OS ± M65 (P value = <0.001). M30 and nDNA were without significant relationship.

The M65 data suggest a potential utility for this marker in prognosis. Further evaluation of the clinical relevance of these findings is warranted with access to a larger patient dataset. The ongoing phase III clinical trials for zibotentan in HRPC patients will provide this opportunity.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C9.
Abstract C92: DNA methylating combi-molecule ZRS1 induces p53 and blocks the epidermal growth factor (EGFR)-activated pathways in EGFR-expressing human tumor cells: Relationship to O6-methylguanine DNA methyltransferase (MGMT) status
Authors: Huang Ying , Rachid Zakaria , Qiu Qiyu , Na Ning , Jean-Claude Bertrand J.
Published: June 2012

The combi-targeting concept seeks to design single molecules that combine an EGFR tyrosine kinase inhibitor with a DNA damaging agent. One such molecule, ZRS1 contains an EGFR tyrosine kinase targeting quinazoline moiety and a methyltriazene-based DNA-damaging function capped with an acetoxymethyloxy group that stabilizes the 1,2,3-triazene chain. Here, we analyzed the DNA damage response, the MAPK and the PI3K pathways in response to the dual action of ZRS1. We also determined whether its mixed EGFR-DNA targeting property could enhance its potency in tumor cells expressing the O6-methylguanine DNA methyltransferase (MGMT), a DNA repair enzyme that confers resistance to methylating agents. The growth inhibitory potency of ZRS1 was tested in a panel of 8 human tumor cell lines with varied levels of EGFR and MGMT. ZRS1 was more potent than the clinical methylating agent temozolomide (TEM) in all the cell lines tested, including the lung cancer (A549, A427 and Calu-1), colon cancer [HT29, HCT116 and HCT116 (p53−/−)], and breast cancer cell lines (MDA-MB-468 and MDA-MB-231), regardless of their MGMT status. However, its potency was in the same range as or less than ZD1839, a single-targeted EGFR inhibitor, against MGMT-proficient cells. In the MGMT-deficient cells or MGMT-proficient cells in which MGMT levels were depleted with O6-benzylguanine, its potency was superior to that of ZD1839 or TEM. ZRS1 exhibited IC50 of 0.5 µM in the A427 cell line, a value that was 25-fold lower than that of TEM, 12-fold lower that of ZD1839 and 15-fold lower than that equi-toxic and equimolar combinations of ZD1839+TEM. The two lung cancer cell lines A549 (MGMT+) and A427 (MGMT−) were selected for analyzing the cell signaling pathways underlying the growth inhibitory effect induced by the dual effects of ZRS1. The results showed that: 1. ZRS1 strongly inhibited EGFR phosphorylation in both cell lines and this translated into inhibition of ERK1/2, AKT, Bad S112 and Bad S136 phosphorylation in both cell lines 2. ZRS1 inflicted significant levels of genomic DNA damage and induced p53 in a dose-dependent manner in both cell lines. Likewise Bax and GADD45 were also induced in both cell lines. The results in toto suggest that in the presence of MGMT, ZRS1 primarily acts as an EGFR inhibitor with no significant effect of its DNA damaging component. In contrast, in the absence of MGMT, its ability to (a) activate the p53 pathway (b) block the MAPK and PI3K pathways, translates into significant cell-killing. Further studies with plasmid transfection either to knockdown endogenous MGMT or express exogenous MGMT are ongoing to elucidate the mechanism underlying the suppression of the potency of ZRS1 by MGMT.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C92.
Abstract A100: Pemetrexed-induced uracil accumulation is an importatn source of DNA damage in UNG deficient cells, contributing to hematologic toxicity.
Authors: Weeks Lachelle D. , Zetner Gabriel , Scacheri Peter , Liu Lili , Gerson Stanton L.
Published: June 2012

Pemetrexed is a multi-target antifolate that has proven activity against several solid tumors. Dose limiting myelosuppression and acquired resistance are major challenges to pemetrexed and antifolate chemotherapy regimens. We have previously shown that resistance to pemetrexed in human cancer cells can be overcome via inhibition of DNA base excision repair (BER), the major pathway responsible for the resolution of uracil in DNA. To understand the implications of UNG deficiency in vivo, we have investigated the impact of UNG deficiency on whole animal response to pemetrexed. Here, we illustrate that pemetrexed treatment induces more significant reduction in bone marrow cell numbers and relatively low peripheral lymphocyte counts in UNG−/− mice compared to UNG+/+ littermates. Compared to wild-type cells, UNG−/− MEFs also show reduced viability and increased cell death in response to pemetrexed. Hematopoietic cell sensitivity was mirrored by increased pemetrexed cell killing in UNG mutant human lymphoblastoid cells, derived from patients with biallelic inactivating mutations in the UNG gene. Cellular sensitivity is characterized by reduced nucleotide incorporation, arrest in S-phase of the cell cycle, and induction of apoptosis. Pemetrexed treatment of UNG−/− cells also causes robust induction of γ-H2AX, a surrogate marker of DNA double strand breaks. Through γ-H2AX ChIP-sequencing we provide the first direct evidence that DNA double strand breaks are located specifically at AT(U) rich sequences, directly implicating uracil accumulation in the formation of double strand breaks following pemetrexed treatment. UNG deficient cells showed no differential sensitivity to cisplatin or temozolomide, two drugs unrelated to uracil accumulation. Moreover, through thymidine supplementation, we were able to partially reverse the pemetrexed-mediated induction of DNA damage markers, including γ-H2AX and protect UNG−/− cells from cell death, further supporting a uracil-mediated mechanism of cytotoxicity in UNG−/− cells. In sum, these data suggest that the accumulation of uracil in the DNA of hematopoietic cells drives myelosuppression and lymphocyte toxicity. Mechanistically, our data support a previously underappreciated role for uracil accumulation, UNG activity and the BER pathway in the determination of cellular sensitivity to pemetrexed and suggest UNG expression may be an important predictor of adverse events, such as myelosuppression in individuals receiving pemetrexed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A100.
Abstract A104: Removal of uracil by uracil DNA glycosylase limits pemetrexed cytotoxicity: Overriding the limit with methoxyamine (TRC102) to inhibit base excision repair.
Authors: Bulgar Alina , Weeks Lachelle D. , Miao Yanling , Yang Shuming , Xu Yan , Guo Chunguang , Markowitz Sanford , Gerson Stanton L. , Liu Lili
Published: June 2012

Uracil DNA glycosylase (UDG) is a conserved DNA repair protein existing in all types of human cells. Since UDG specifically removes uracil bases from DNA, its repair activity determines cell sensitivity to anticancer agents that are capable of introducing uracil into DNA. We studied the participation of UDG in response to pemetrexed-induced incorporation of uracil into DNA using isogenic human tumor cell lines with or without UDG (UDG+/+/UDG−/−). Results revealed that UDG−/− cells were very sensitive to pemetrexed. Cell killing by pemetrexed was associated with genomic uracil accumulation, stalled DNA replication, and catastrophic DNA strand breaks. In contrast, UDG+/+ cells were >10 times more resistant to pemetrexed due to a rapid removal of uracil from DNA, generating abasic sites (AP sites). Resultant AP sites were efficiently repaired by base excision repair (BER) so that they contributed little to cell death. However, addition of the BER inhibitor methoxyamine (TRC102) synergistically enhanced the cytotoxicity of pemetrexed in UDG proficient cells. MX bound covalently to AP sites to form unrepairable MX-bound AP sites, thereby inhibiting the BER pathway. Importantly, we identified that MX-bound AP sites induced cell death by inactivating UDG and poisoning topoisomerase II. These two genes are highly expressed in lung cancer cells in comparison with normal bone marrow cells. Thus, targeting UDG/BER to enhance pemetrexed cytotoxic effects is a target-based strategy that selectively mediates cytotoxicity on cancer cells through a synthetic lethal mechanism.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A104.
Abstract A106: Investigation of 5-fluorodeoxycytidine with tetrahydrouracil as a demethylation regimen in solid tumors.
Authors: Kinders Robert J. , Wang Lihua , Kummar Shivaani , Khin Sonny , Balasubramanian Priya , Zhu Weimin , Parchment Ralph E. , Newman Edward , Tomaszewski Joseph E. , Doroshow James H.
Published: June 2012

Methylation-induced silencing of gene promoters is widely documented in numerous cancer types, and the anti-tumor activity of agents that target this phenomenon has established DNA methyltransferase (DNMT1) as a valid drug target. We have investigated the combination of 5-fluorodeoxycytidine with Tetrahydrouridine (FdCyd + THU) in vitro and in vivo using continuous exposures up to 12 weeks. Supplementation of the medium with Deoxythymidine (10 μM) was required to prevent FdCyd cytotoxicity and allow sufficient time for demethylation to occur. Decitabine was used as the positive control throughout the evaluation. The bladder cancer line EJ6 was sensitive to FdCyd + THU, as evidenced by an increase in cell doubling time, a decrease in LINE1 methylation, and an initial decrease in total DNMT1 concentrations, determined with a newly-developed, quantitative two-site immunoassay. The triple negative breast cancer line MDA-MB-231 was resistant to the regimen, as assessed by all three criteria. The mechanism of this combination regimen in tumor cells isolated from paracentesis specimens from patients enrolled on an NCI-supported Phase 1 clinical trial of FdCyd + THU; and was confirmed by finding changes in both DNMT1 levels and LINE1 methylation. Upregulation of expression of p16 (INK4a), but not GSTP1 or RASSF1, was observed in EJ6 cells following FdCyd + THU by Western blotting. An immunofluorescence assay for p16 expression in circulating tumor cells (CTCs) has been developed and implemented in the Phase 1 trial. The ongoing Phase 2 trial of this regimen will include measurements of DNMT1 and LINE1 methylation in tumor biopsies and p16 in CTCs to directly assess drug effect on the DNMT1 target.

This Research has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E and NCI RAID Project #266 Grant.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A106.
Abstract A13: Characterization of CRC primary explant models in comparison to standard human tumor cell-line-derived CRC xenografts.
Authors: Alferez Denis G. , Brown Helen , Dry Jonathan R. , Runswick Sarah , James Neil , McWalter Gael , Whiteley Jessica , Revill Mitch , Beran Garry , Farren Matthew , Smith Neil R. , Hedge Phillip , Barry Simon , Wedge Stephen R. , Wilkinson Robert W.
Published: June 2012

Primary tumor xenograft models (PTX) are established in immunodeficient mice by implantation of material directly from patient tumors. These models are propagated in vivo so that they are not subjected to additional selection pressures within tissue culture. The models reputedly reflect a greater genetic diversity of disease than can be recapitulated within tumor cell line derived xenografts and may have morphological differences. Consequently, such models may be of greater relevance for the evaluation of drug efficacy.

To verify differences versus cell line derived xenograft models, we have examined samples of 42 CRC PTX models, derived from Dukes stage A-D tumor samples, which were obtained from European contract research organisations) Characterisation included analysis of common genetic mutations (KRas, BRAF, PI3Ka, PTEN, P53 and APC), mRNA expression via microarray platforms (Affymetrics HG_U133_plus_2), high-throughput RT-PCR of specific probes (mouse and human) to examine stromal genes, and the activation of particular proteins using reverse phase antibody arrays and immunohistochemistry. Comparisons were made with 6 commonly used CRC cell line derived xenografts.

The PTX models overall represent a broader range of molecular pathology - for example the inclusion of wild-type Kras tumors. Histopathological characterisation also confirmed that the PTX models have a higher stromal content that is routinely observed in cell line derived xenografts, and a more complex morphology with higher tumor cell differentiation.

PTX models may potentially complement drug discovery activities by providing a wider platform in which to test preclinical hypotheses - based upon a defined biological mechanism and/or a genetic determinant of sensitivity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A13.
Abstract A147: EZN-3920, a HER3 antagonist, enhances the antitumor effect of gefitinib and lapatinib in xenograft models.
Authors: Wu Yaming , Li Qi , Wang Maoliang , Qu Zhengxing , Kim Steven , Zhang Yixian , Greenberger Lee M.
Published: June 2012

Background: The HER family consists of four tyrosine kinase receptors designated as EGFR, HER2, HER3 and HER4. Numerous new studies now indicated that HER3 plays a critical role in cancer development since it 1) heterodimerizes with EGFR or HER2, 2) is a critical link to the PI3K/AKT axis, and 3) can mediate resistance to HER1/2-targeted therapeutics. Resistance to the tyrosine kinase inhibitors (TKIs), such as gefitinib and lapatinib, are associated with overexpression of HER3 ultimately leading to activation of the PI3K/AKT axis. Thus, it is a logical approach to combine agents capable of down modulating HER3 with TKIs. To this end, we have used an RNA antagonist of HER3, EZN-3920, to explore its ability to inhibit tumor growth and enhance the activity of HER1/2-directed therapeutics.

Methods: Tissue culture cells were treated with EZN-3920, a locked nucleic acid (LNA)-based oligonucleotide complementary to HER3. EZN-3920 was either added to tissue culture media (i.e. no transfection) in vitro or prepared in saline and given IV in vivo. Endpoints were measured by qRT-PCR, MTT, Western Blot analysis, Immunohistochemistry, and tumor size.

Results: EZN-3920 down modulated HER3 mRNA, protein expression, PI3K/AKT signaling, and inhibited tumor cell proliferation. In vivo systemic administration of EZN-3920 resulted in specific down-modulation of HER3 mRNA and protein expression, as well as blockade in PI3K/AKT signaling pathways in tumors derived from HCC827 lung carcinoma cells that was associated with tumor growth inhibition. Interestingly, the combination of EZN-3920 at 30 mg/kg (biweekly for 4 weeks, i.v.) and gefitinib at 15 mg/kg (5 times a week for two weeks, p.o) completely shrank established HCC827 tumors while either treatment alone only delayed tumor growth by 40–60%. Similarly, in BT-474-M1 breast carcinoma xenograft models, EZN-3920 in combination with lapatinib resulted in superior antitumor effect than either agent alone.

Conclusions: The studies suggest that the antitumor effects of gefitinib or lapatinib can be enhanced by HER3 down modulation in NSCLC and breast tumors, respectively. On-going studies will determine if tumors that are resistant to HER1/2 therapeutics will still respond to EZN-3920 alone or its combination with standard HER1/2 therapeutics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A147.