bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B69: Enhancement of nonhomologous end joining DNA repair capacity confers cancer cells resistant to novel selenophene compound D-501036
Authors: Yang Yung-Ning , Pan Wen-Yu , Yeh Ssu-Ching , Tsou Tsui-Chun , Chen Li-Tzong , Chang Jang-Yang
Published: June 2012

D-501036 is a selenophene compound, which has been shown to exhibit antineoplastic activity against various types of tumor. Our pervious data demonstrated that the treatment of D-501036 induces S phase arrest during cell cycle. It induces the formation of Se-DNA adducts and reactive oxygen species (ROS) in cells. Moreover, the amount of signal transduction proteins responded to DNA damage such as phospho-ATM and phospho-p53 were increased in D-501036-treated cells (Mol Cancer Ther, 2007). To investigate the mechanism of resistance to selenophene compounds, a human cervical cancer KB-derived D-501036-resistant KB-1036-S4 cells were generated. This D-501036-resistant cancer cell lines also exhibits cross-resistance to cisplatin, oxaliplatin and CPT compounds. Although the treatment of D-501036 induced similar level of ROS and Se-DNA adducts in both drug-sensitive (KB) and -resistant (KB-1036-S4) cells, DNA was less damaged in KB-1036-S4 as compared to KB cells. Decrement of DNA damage in drug-resistant cells indicates that the efficiency of DNA repair during genomic stresses might be higher in KB-1036-S4 cells. Pervious studies suggested that D-501036 induces double strand DNA break (DSB). Global DNA repair ability in KB- 1036-S4 cells is higher than KB cells. In vitro Non-homologous end joining (NHEJ) assay with the use of whole cell lysate revealed that KB-1036-S4 also exhibits higher ability of forming DNA multimer by rejoining linear DNA strand. To determine the importance of NHEJ in drug resistance of KB-1036-S4 cells, ku80 was down-regulated by siRNA. Ku80 is a major component in NHEJ, which is responsible for the recruitment of XRCC4 and DNA ligase IV. Cell viability assay revealed that gene silencing of Ku80 by siRNA partially restored sensitivity to D-501036 in KB-1036-S4 cells. The IC50 of D-501036 in Ku80-downregulated KB-1036-S4 cells was reduced to 25% as compare to negative control. Global DNA repair ability of KB-1036-S4 was also decreased in Ku80-downregulated situations. Taken together, these results indicate that enhancement of NHEJ activity is one of the major resistance mechanisms to D-501036. Targeting components in the NHEJ pathway seems to be a promising way to overcome resistance to DNA damaging agents.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B69.
Abstract B74: MET activation and Cetuximab resistance in patient-derived tumor xenografts
Authors: Krumbach Rebekka , Schüler Julia , Hofmann Michael , Fiebig Heinz-Herbert , Beckers Thomas
Published: June 2012

Cetuximab (Erbitux), by targeting the epidermal growth factor receptor (EGFR), strongly inhibits tumor growth and is approved for the treatment of colon as well as head and neck cancer. Although expression of the EGFR is evident in a multitude of tumor histotypes, strong responses to therapy are only seen in selected patients and histotypes. In the present study we assessed the responsiveness to Cetuximab therapy given at 30mg/kg, qd7x3 and explored putative resistance mechanisms in 34 patient-derived xenografts established at Oncotest representing NSCLC, head and neck, gastric, colorectal and mammary cancer as well as melanoma.

With a threshold of 35% as best T/C activity in vivo, 27 out of 34 tumor models (79.4%) were Cetuximab resistant despite strong inhibition of EGFR phosphorylation in tumors of treated animals. The mutational status for Kiras and B-raf was analyzed by exon sequencing with 10 and 4 out of 34 tumor models having Ki-Ras and B-Raf mutations, respectively. While all B-raf mutant tumors were resistant towards Cetuximab treatment, this was true for 8 out of 10 Ki-ras mutant tumors. As to EGFR family members, HER2 was highly expressed and activated only in the mammary adenocarcinoma MAXF1162, whereas phosphorylated HER3 was identified more broadly in Cetuximab-sensitive and resistant tumors. Interestingly, c-Met was highly activated only in the resistant LXFA526 and LXFA1647 lung adenocarcinoma. The function of c-Met was studied in more detail by siRNA-induced gene knock-down using the respective cell lines LXFA1647L and LXFA526L. In both lung adenocarcinoma, activation of c-Met was essential for anchorage-independent growth as analyzed using the tumor colony formation assay. Interestingly, LXFA526L cells with c-Met knock-down displayed EGF-dependent colony growth. As a further consequence of c-Met downregulation, EGFR phosphorylation was strongly reduced, indicating a functional interaction of c-Met and EGFR in LXFA526L cells, supported by co-immunoprecipitation of both receptors.

In summary, constitutive activation of c-Met which is essential for oncogenic transformation in selected lung adenocarcinoma is a mechanism of primary Cetuximab resistance. Constitutively activated receptors like EGFR can support anchorage-independent, tumorigenic growth in vivo when c-Met is inhibited. Therefore, a combination therapy with c-Met and EGFR inhibitors (Cetuximab or a small molecule kinase inhibitor like Erlotinib) potentially have additive or even synergistic anti-tumor activity in c-Met and EGFR expressing NSCLC patients.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B74.
Abstract B86: HSP90 is a promising target in gemcitabine and 5-fluorouracil resistant pancreatic cancer
Authors: Vashist Yogesh K. , Kalinina Tatyana , Rabofski Judith L. , Gebauer Florian , Mina Sormeh , Guengor Cenap , Izbicki Jakob R. , Yekebas Emre F.
Published: June 2012

Purpose: Pancreatic cancer (PC) is a deadly disease. Chemotherapy options are limited to gemcitabine and 5-fluorouracil (5-FU). The molecular profile of PC offers several growth receptors (epidermal growth factor receptor [EGFR], insulin-like growth factor 1 receptor [IGF-1R]) and tyrosine kinases (AKT and MAPK) as promising therapy targets. EGFR, IGF-1R, AKT and MAPK are dependent upon the heat shock protein 90 (HSP90) to gain the mature functional active protein form hence HSP90 represents a promising therapy target in PC. Functional inhibition of HSP90 has the advantage of disruption of many oncogenic signalling pathways simultaneously. The aim of this study was evaluate the efficacy of different HSP90 inhibitors in gemcitabine and 5-FU resistant PC.

Material and Methods: Human gemcitabine and 5-FU resistant pancreatic cancer cell lines 5061, 5072 and 5156 were generated and brought in to culture from patients who were operated for PC at our institution and received gemcitabine as adjuvant therapy. The commercial L3.6pl cell line served as a control. Antiproliferative efficacy of three different HSP90 inhibitors (17AAG, 17DMAG and 17AEPGA) was evaluated by the MTT assay. Alterations in signalling pathway effectors and apoptosis upon HSP90 inhibition was determined by western blot analysis (WB).

Results: The cell lines 5061, 5072 and 5156 were resistant to gemcitabine and 5-FU. In contrast, 17AAG and the water-soluble derivates 17DMAG and 17AEPGA displayed high antiproliferative activity in all tested cell lines. The calculated GI50 was below 1 µM. In line with the proliferation inhibition in the MTT assay, the expression of EGFR, IGF-1R, AKT and MAPK was significantly down regulated under HSP90 inhibition as verified inWB. In contrast, HSP70 and HSP27 were upregulated in all cell lines under HSP90 inhibition. Apoptosis was an early event in all three HSP90 inhibited cell lines and determined by CASPASE-3 and poly (ADP-ribose) polymerase assay. In contrast, gemcitabine treated cells did not show significant apoptosis.

Conclusions: Functional inhibition of HSP90 disrupts multiple signalling cascades in gemcitabine and 5-FU resistant PC. Water-soluble 17DMAG is equally effective as 17AAG. The up-regulation of HSP70 and HSP27 indicates counter regulation of tumor cells to prevent apoptosis and should be addressed when considering HSP90 inhibition in PC.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B86.
Abstract B93: Sensitivity to the MEK inhibitor, E6201, is associated with mutant BRAF status and is inversely correlated with pAKT expression levels in melanoma cell lines
Authors: Byron Sara , Wellens Candice , Wu Jiayi , Wang John , Nomoto Kenichi , Pollock Pamela
Published: June 2012

Inhibition of the RAS/RAF/MEK/ERK signaling pathway has emerged as a promising therapeutic strategy for melanoma given the high frequency of BRAF and NRAS mutations in this tumor type. Sensitivity to E6201, an inhibitor of MEK1 and other cancer relevant kinases, was assessed in a panel of 32 cell lines for which the mutation status of common melanoma genes was known. The majority (24/32) of the melanoma cell lines were sensitive to E6201, resulting in G1-cell cycle arrest and cell death in 11 sensitive cell lines and cell cycle arrest but not cell death in two sensitive lines. Xenograft studies revealed that E6201 exhibited a cytotoxic effect in vivo, even in cell lines where only a cytostatic effect was observed in vitro. Among cell lines carrying mutations in BRAF, or NRAS, sensitivity to E6201 was associated with wildtype PTEN status (p<0.03). When the cell lines were classified based on basal pERK levels rather than BRAF mutation status, high levels of pAKT correlated with insensitivity to E6201 (p<0.04). Indeed, high levels of pAKT correlated with E6201 insensitivity independent of BRAF or PTEN mutation status (p<0.03). Together these data demonstrate the cytostatic and cytotoxic activity of E6201 in BRAF mutant melanoma cell lines in vitro and in vivo and, furthermore, indicate that quantification of pAKT levels may have clinical utility as a biomarker of E6201 resistance.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B93.
Abstract B96: Akt and c-Myc differentially activate cellular metabolic programs and prime cells to bioenergetic inhibition
Authors: Fan Yongjun , Dickman Kathleen G. , Zong Wei-Xing
Published: June 2012

The high glucose consumption of tumor cells even in oxygen rich environments, referred to as the Warburg effect, has been noted as a nearly universal biochemical characteristic of cancer cells. Targeting the glycolysis pathway has been explored as an anti-cancer therapeutic strategy to eradicate cancer based on this fundamental biochemical property of cancer cells. Oncogenes such as Akt and c-Myc regulate cell metabolisms. Accumulating studies have uncovered various molecular mechanisms by which oncoproteins affect cellular metabolism, raising a concern as to whether targeting glycolysis will be equally effective in treating cancers arisen from different oncogenic activities. To address this issue, we establish a dual-regulatable FL5.12 pre-B cell line in which myrAkt is expressed under the control of doxycycline, and c-Myc, fused to the hormone-binding domain of the human estrogen-receptor (ER), is activated by tamoxifen. Using this system we directly compare the effect of these oncoproteins on cell metabolism in an isogenic background. Activation of either Akt or c-Myc leads to the Warburg effect as indicated by increased cellular glucose uptake, glycolysis, and lactate generation. When cells are treated with glycolysis inhibitors, Akt sensitizes cells to apoptosis whereas c-Myc does not. In contrast, c-Myc but not Akt sensitizes cells to the inhibition of mitochondrial function. This is correlated with enhanced mitochondrial activities in c-Myc cells. Hence, while both Akt and c-Myc promote aerobic glycolysis, they differentially affect mitochondrial functions and render cells susceptible to the perturbation of cellular metabolic programs.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B96.
Abstract C106: Discovery and characterization of INCB024360, a potent and selective inhibitor of indoleamine 2,3-dioxygenase (IDO1) as a novel agent for cancer immunotherapy
Authors: Liu Xiangdong , Shin Niu , Koblish Holly , Yang Gengjie , Wang Qian , Wang Kathy , Leffet Lynn , Thomas Beth , Rupar Mark , Waeltz Paul , Bowman Kevin , Sparks Richard , Yue Eddy , Li Yanlong , Wynn Richard , Fridman Jordan , Burn Timothy , Combs Andrew , Newton Robert , Scherle Peggy
Published: June 2012

Indoleamine 2,3-dioxygenase (IDO or IDO1) mediates the oxidation of tryptophan, an amino acid essential for cell proliferation and survival. Inhibition of IDO activity or expression has shown therapeutic potential in preclinical models of immunodeficiency-associated abnormalities including cancer. Here we report the identification of INCB024360, a novel, potent and selective small molecule inhibitor of IDO1, and the investigation of its effects on the proliferation and activation of various immune cells in vitro as well as its anti-tumor activity in preclinical tumor models. In multiple cell-based assays, INCB024360 potently inhibits human IDO1 with an IC50 of approximately 10 nM - the most potent IDO1 inhibitor published thus far. INCB024360 is IDO1 selective and has little activity against other related enzymes including IDO2, TDO or tryptophan transporters. In co-culture systems of human allogeneic lymphocytes with either dendritic cells or tumor cells, inhibition of IDO1 by INCB024360 increases the proliferation of T and NK cells, as well as IFN- production. INCB024360 addition also reduces the generation of regulatory T cells. Interestingly, IDO1 expression promotes dendritic cell apoptosis while addition of INCB024360 reverses this and increases the number of CD86high cells within the IDO+ DC population, potentially representing a novel mechanism by which IDO1 inhibition can promote T cell activation. In vivo, INCB024360 inhibits tryptophan catabolism in tumors and tumor draining lymph nodes and controls tumor growth either alone or in combination with chemotherapeutic agents in multiple tumor models. The ability to reduce tumor growth is dependent on a functional immune system, consistent with the proposed mechanism of action. Finally, an analysis of kynurenine/tryptophan levels in patient blood samples affirms that the IDO activity is elevated in multiple tumor types. Collectively, these data suggest that INCB024360, a potent IDO1 selective inhibitor, has the potential to be a novel and effective immunotherapeutic agent for cancer treatment.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C106.
Abstract C112: Pharmacokinetically guided phase 1 trial of the IGF-1 receptor antagonist R1507 administered weekly in children with recurrent or refractory solid tumors
Authors: Bagatell Rochelle , Herzog Cynthia E. , Trippett Tanya M. , Winter Erica , Grippo Joseph F. , Fox Elizabeth , Yurasov Sergey , Balis Frank M. , Gore Lia
Published: June 2012

R1507 is a fully human monoclonal antibody that binds the IGF-1 receptor (IGF1R) and blocks IGF1R-mediated signal transduction. A phase 1 trial of weekly R1507 in adults did not define a maximum tolerated dose (MTD), and dose-limiting toxicity (DLT) was not observed. This pediatric phase 1 trial of R1507 was designed to identify a dose that achieves a mean area under the concentration x time curve (AUC0–7d) >85% of the mean AUC0–7d (18,700 mcg•h/mL) in adults at the recommended phase 2 dose (RPTD) of 9 mg/kg weekly. The design can define an MTD if DLT occurs. Patients (pts) age >2 and <18 years (y) with relapsed or refractory solid tumors including brain tumors were eligible. Detailed PK sampling was performed after the 1st dose; selected peak and trough levels were obtained to week 10. Nineteen evaluable pts (median age 10 y, range 3–17) with Ewing sarcoma (3), rhabdomyosarcoma (4), adrenocortical carcinoma (2), ependymoma (2), liver tumors (4), or other solid tumors (4) were enrolled. The AUC0-7d (mean 5,670 mcg•h/mL; CV 30%) at the starting dose of 3 mg/kg (n=3) was below the target AUC0-7d, but the AUC0-7d in the first 3 pts treated with 9 mg/kg (mean 22,500 mcg•h/mL) exceeded the target. This cohort was expanded to study 3 age groups: 2–6, 7–11, and 12–17 y. The mean AUC0-7d in the expanded cohort (n=16) was 21,500 mcg•h/mL (CV 25%), but was lower in the 2–6 y cohort (Table) where 2/4 pts did not achieve the target AUC0-7d. In simulations, 300 mg/m2 dosing yielded more uniform drug exposure across the age range (Table). The half-life at 9 mg/kg was 4–8 days. The mean trough concentration on day 35 was 224 mcg/mL. Accumulation was modest (trough35d:trough7d= 2.7). DLT and cumulative toxicity were not observed over a median of 5 weeks of treatment (range 1–44). Toxicities included fatigue, nausea, arthralgia, extremity pain, and anorexia; all were grade <3. Six pts had stable disease ≥12 weeks' duration, including 2 with Ewing's sarcoma and 2 with ependymoma. The recommended R1507 dose is 9 mg/kg weekly in children age > 6 years. Dosing based on body surface area may be preferable for children <6 y.

Single Dose PK Parameter Mean (%CV) Age group Cmax (mcg/mL) CL (mL/day/kg) AUC0–7d (mcg·h/mL) Ctrough, steady state (day 35) (mcg/mL) Simulated AUC0–7d (mcg·h/mL)* 2–6 y 222 (20) 7.1 (15) 17,100 (13) 196 24,800 (n=4) (N/A n=1) 7–11 y 253 (30) 7.0 (27) 20,900 (30) 208 23,000 (n=6) (39; n=5) 12–17 y 293 (16) 5.5 (28) 25,000 (16) 248 24,400 (n=6) (39; n=5) * Simulated AUC0–7d for dosing with 300 mg/m2

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C112.
Abstract C122: Clinical implication of ERCC1 overexpression in advanced biliary tract adenocarcinoma patients treated with cisplatin-based palliative chemotherapy
Authors: Hwang In Gyu , Lee Gyeong-Won , Kang Jung Hun , Oh Sung Yong , Kwon Hyuk-Chan , Jun Hyun Jung , Lim Ho Yeong , Lee Seungkoo , Jang Joung-Soon , Lee Sang-Jae
Published: June 2012

Several clinical studies have shown that excision repair cross-complementation group1 (ERCC1) overexpression is associated with resistance to platinum-based chemotherapy and poor prognoses in several tumors. However these studies have never been tried for biliary tract cancer.The aim of this study was to evaluate the significance of ERCC1 expression as a predictive marker, we analyzed the association between ERCC1 expression and treatment outcomes in advanced biliary adenocarcinoma patients treated with cisplatin-based palliative chemotherapy. 74 patients with histologically confirmed adenocarcinoma of biliary tract were enrolled at 5 institutions between January 2002 and August 2008. Of 74 patients, 65 was assessed by immunohistochemistry from biopsy specimens. ERCC1 expression was positive in 33 out of 65 specimens (51%). 30 (46.2%) of 65 patients showed clinical benefits(including complete response, partial response and stable disease). Among 30 patients who obtained clinical benefits, eleven (37%) were positive for ERCC1 expression and nineteen (63%) were negative (p=0.048). With a median follow-up of 7.3 months (range, 0.8–47.5 months), median progression free survival (PFS) and overall survival (OS) were 3.0 months (95% CI, 1.3–4.7 months) and 7.8 months (95% CI, 5.9–9.7 months), for the total study population. Among patients treated with cisplatin, those who had ERCC1-negative tumors showed longer survival. On univariate analysis, PFS was 1.9 months for patients with ERCC1 positive tumor and 4.6 months for those with ERCC1 negative tumor (P = 0.014). OS was significantly longer in the ERCC1-negative group than in the ERCC1-positive group (9.1 vs 7.3 months, respectively; P = 0.017). A Cox proportional hazard model showed that negative ERCC1 expression (hazard ratio 0.401, 95% CI, 0.176–0.912; P = 0.029), clinical response (hazard ratio 0.290, 95% CI 0.120–0.705; P = 0.006), peritoneal metastasis (hazard ratio 0.260, 95% CI 0.082–0.828; P = 0.023) and primary cancer site (hazard ratio 0.228, 95% CI 0.064–0.814; P = 0.023) were significant independent prognostic factors for the prolongation of OS. These results suggest that advanced biliary tract adenocarcinoma patients with ERCC1-negative tumors show both clinical benefit and survial benefit from palliative chemotherapy with a cisplatin-containing regimen.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C122.
Abstract C142: Safety of single-walled carbon nanotubes for delivery of siRNA
Authors: Kirkpatrick Lynn , Bartholomeusz Geoffrey , Weiss Michelle , Tsyboulski Dima , Ghosh Saunab , Weisman Bruce
Published: June 2012

siRNA is a leading potential new cancer therapy but currently lacks an adequate delivery mechanism. Ensysce has been exploring the use of single-walled carbon nanotubes (SWCNT) as delivery vehicles for siRNA delivery and has previously reported on the in vitro and in vivo efficacy of this platform. This study was conducted to determine what if any toxicities result from the use of SWCNT and identify toxicities that may be directly attributed to this delivery vehicle. For this application, it is not only necessary to determine the maximal load of SWCNT that can be tolerated, the dose limiting toxicities and the fate of the material in the body, but also to carefully prepare the SWCNT suspensions to ensure that contaminating metals, nanotube aggregates and other materials are not present when administered systemically. Ensysce Biosciences has been exploring the safe use of this material and here presents its latest findings from a toxicology study where the total dose of 50 and 100 g of fully suspended and individualized SWCNT were administered to groups of 3 mice. Mice were sacrificed at 12, 24, 48 hr and 1 week following i.v. administration. Blood chemistry and hematology were evaluated at each time point. Urine and feces were collected at 12, and 24 hr. Organs, including spleen, liver, kidney, heart, lung and brain were examined macroscopically and by near IR-fluorescence for the presence of SWCNT. The presence and persistence of SWCNT in the tissues was measured. Data to be presented will include details of the analytical characterization of the SWCNT preparations that were delivered, showing that the tubes were well dispersed and individualized. At both the 24 hr and 1 week timepoints, blood chemistry and hematology showed no evidence of toxicity, as values for all were within the normal ranges. Macroscopic examination of organs indicated that SWCNT were present in liver and spleen at 24 hr. Near IR-fluorescence spectroscopy demonstrated that the nanotube accumulation in the spleen decreased over a 1 week period. Data will be presented showing that a 100 g dose (equivalent to 30 mg in humans) was well tolerated and non-toxic. These studies have led to further work examining limits for use of SWCNT for siRNA delivery, including stability of the siRNA complexes in serum, siRNA loading efficiency and systemic efficacy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C142.
Abstract C167: Trop2 as a target for breast cancer treatment
Authors: Wang Jianbo , Day Ryan , Dong Yiyu , Weintraub Steven , Michel Loren
Published: June 2012

Trop2 is expressed on many epithelial tumors and stem cells of the prostate and liver. Its association with aggressive cancer has led to the speculation that it might be a therapeutic target. We have previously shown a role for Trop2 in colon tumorigenesis and also found that antibodies against Trop2 can block tumor cell invasion. Consistent with this data, a monoclonal antibody against Trop2 is in pre-clinical development against several tumor types. However, the expression pattern and role of Trop2 in breast cancer has not been examined. Here, we show heterogeneous expression of Trop2 in normal mammary gland, primary breast tumors and cancer cells lines. After purification of Trop2 expressing tumor cell populations by fluorescence activated cell sorting, we find that Trop2hi cells are preferentially invasive and tumorigenic compared to the Trop2lo. RNAi mediated suppression of Trop2 in breast cancer cells arrests their proliferation. Even more importantly, a monoclonal antibody against Trop2 shows anti-tumor activity in vivo. Therefore, Trop2 represents a novel and unexplored therapeutic target in this common malignancy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C167.
Abstract C173: Anti-tumor activity of SCH 900105 (AV299), an anti-HGF antibody, in non-small cell lung cancer models
Authors: Meetze Kristan A. , Boudrow Andrea , Connolly Kelly , Huang Riyun , Rideout William , Gyuris Jeno , Han May
Published: June 2012

Hepatocyte growth factor (HGF) is the soluble ligand for the c-Met receptor tyrosine kinase. Signaling through the HGF/c-Met pathway mediates a plethora of cellular activities that are involved in cancer cell dysregulation, tumorigenesis, and metastasis including cell proliferation and survival, angiogenesis, migration, invasion and drug resistance. HGF/c-Met autocrine and paracrine regulatory loops have been reported in a number of non-small cell lung cancer studies. Furthermore, studies have shown that HGF/c-Met pathway upregulation via either c-Met amplification or HGF secretion can result in intrinsic or acquired resistance to EGFR TKIs in lung adenocarcinoma.

SCH 900105, formerly known as AV-299, is a humanized IgG1 antibody with high affinity to HGF that neutralizes all its biological functions tested with sub-nM potency. It is currently in phase 1 clinical trials that demonstrated good safety and tolerability. Anti-tumor activity of SCH 900105 was observed in HGF autocrine and paracrine in vivo tumor models, such as GMB, pancreatic cancer and multiple myeloma.

Anti-tumor efficacy of SCH 900105 was evaluated in paracrine models of the HGF-dependent NCI-H596 NSCLC cell line xenografted in SCID mice engineered to produce human HGF. In these models, SCH 900105 treatment resulted in dose-dependent decrease in tumor growth with concurrent increases in serum and tumor concentration of SCH900105 and increases in serum SCH 900105/HGF complex. Treatment also led to significant reduction in phospho-c-Met and phospho-Akt levels in tumor lysates. Concurrently, increases in cleaved caspase-3 and decreases in Ki67 and CD31 staining were also observed. Anti-tumor activity of SCH 900105 was also explored in combination with EGFR inhibitors, erlotinib and Cetuximab that resulted in increased efficacy. SCH900105 treatment resulted in decreased phospho-c-Met levels with concurrent increases in phospho-EGFR levels. Conversely, erlotinib treatment decreased phospho-EGFR with concurrent increases in phospho-Met levels. The combination of SCH900105 with Cetuximab resulted in complete response in all animals treated without tumor re-growth 50 days after treatment withdrawal.

Potent anti-tumor activity of SCH 900105 in combination with EGFR inhibitors observed in these preclinical models suggests testing the combination in NSCLC is warranted in the clinic.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C173.
Abstract C177: Src and ADAM17-mediated shedding of heregulin is a mechanism of acute resistance to chemotherapy in gastro-oesophageal cancer cells
Authors: Campbell Lynn Rachel , Van Schaeybroeck Sandra , Kyula Joan , Eatock Martin , Johnston Patrick
Published: June 2012

Background: We previously reported that, in response to chemotherapy, gastro-oesophageal cancer cells (GEC) activate a human epidermal receptor (HER)-mediated survival response abrogated by HER inhibitors. In order to elucidate potential biomarkers for response to chemotherapy treatment and potential novel targets, we investigated the pathways which mediate chemo-induced HER activity in GEC cells.

Methods: Apoptosis was measured by Flow Cytometry or PARP, Caspase 8/3 cleavage. HER1/HER2/HER3, Akt expression/activity and Heregulin (HRG) expression levels were determined by Western blotting. ADAM17 activity was measured using a fluorometric (MCA) assay kit while sTGF-α levels were analysed by ELISA.

Results: Following treatment with chemotherapy, we found that increased HER1/HER2/HER3 activity was associated with increased Src-family kinase (SFK) activity. Using the SFK inhibitor AZD0530, we found that chemotherapy-induced HER1 activation was abolished, indicating that HER1 activation was mediated via SFKs. Furthermore, we found that the dual HER1/HER2 inhibitor lapatinib had no effect on chemo-induced SFK activity, indicating that SFKs act upstream of HER. Furthermore, we found that chemotherapy treatment resulted in increased ADAM-17 activity, TGF-α and heregulin levels in culture medium of GEC cells. ADAM17 inhibition attenuated chemotherapy-induced TGF-α and heregulin levels and HER1/3 activity. When cells were co-treated with heregulin-neutralizing antibodies, we found strongest inhibition of chemotherapy-induced HER1/HER3 and Akt activity. In addition, following treatment with AZD0530, we found complete inhibition of chemotherapy-induced ADAM17 activity and ligand shedding. Finally, when HER1/HER2/HER3, ADAM17 or SFK inhibitors were combined with chemotherapy, we found a potent increase in apoptosis in GEC cells.

Conclusions: We provide strong evidence that chemotherapy induces HER1/HER2/HER3 activation via a ligand dependent mechanism in GEC cells and have demonstrated that SFK's, ADAM17 and heregulin are critical mediators of HER1/HER2/HER3 activation following chemotherapy. We propose a model in which chemotherapy results in SFK activation that in turn activates ADAM17-mediated shedding of heregulin. Heregulin then activates HER1/HER2/HER3 pro-survival signalling in an autocrine and paracrine fashion. Thus, inhibiting HER1/HER2/HER3, SFKs or ADAM17 may have therapeutic potential for sensitizing GEC tumors to chemotherapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C177.
Abstract C184: Validation of an ELISA for the determination of Rituximab pharmacokinetics in clinical trials subjects
Authors: Ward Tim H. , Hampson Grace , Cummings Jeff , Bayne Mike , Tutt Alison , Cragg Mark , Dive Caroline , Illidge Tim
Published: June 2012

Rituximab is a chimeric anti-CD20 monocolonal antibody directed against the CD20 antigen on the B cell surface. Rituximab was originally developed by IDEC Pharmaceuticals from a high affinity murine monoclonal Antibody (mAb). CD20 is expressed on mature B cells, present on 95% of B cell lymphomas and is thought to act as a calcium channel that regulates progression through the cell cycle. Cross-linking of CD20 by anti-CD20 mAb results in apoptosis through a caspase dependent pathway and may trigger complement dependent cytotoxicity (CDC) or antibody directed cell cytotoxicity. Rituximab has revolutionised the treatment of many B-cell malignancies and is now increasingly being used in non-malignant conditions such as auto-immune disorders. Serum Rituximab levels are highly variable in patients receiving similar ‘standard’ approved doses, and little is known regarding the factors that affect serum Rituximab concentration and that in turn may influence clinical outcome. In order to provide a pharmacokinetic (PK) tool that may ultimately enable patient specific dosing of Rituximab therapy; we have generated a reliable, robust ELISA to quantitate serum Rituximab levels and to provide accurate PK data that will guide the optimisation of rituximab dosing regimes.

An extensive validation of the assay was performed in order to use the assay in the clinical setting. The plate coating efficiency (within and between days) proved reliable resulting in a robust starting platform for the assay. The within day precision for the assay determined using spiked serum samples and was shown to be < 25% with accuracy between 96–109%. The between day precision was <10% with accuracy between 91–125%. Dilution linearity and parallelism were demonstrated with spike recovery for all concentrations and donors within +/− 15% with a coefficient of variation below 10%. The assay has been validated for use on clinical serum samples and consequently stability is a critical factor affecting analysis both in terms of freeze thaw cycles as well as length of storage. To establish sample stability criteria a retrospective study was undertaken using clinical samples collected four years previously as part of a previously published clinical trial. Reanalysed samples showed no statistically significant decrease in measured Rituximab levels compared to the first analysis. These same samples can be freeze thawed and analysed up to five times if necessary.

This assay is highly accurate and reproducible in determining the pharmacokinetic levels of Rituximab in patient serum samples. This assay represents a powerful new tool for clinical evaluation of this widely used therapeutic, it meets stringent acceptance criteria, is fit for purpose, and is currently being applied to several clinical trials incorporating Rituximab in the treatment of Lymphoma.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C184.
Abstract C21: Hypoxia-induced down-regulation of BRCA1 nuclear protein in human breast cancer tissues
Authors: Li Jia , Sullivan Catherine AW. , Numeister Veronique , Rimm David L. , Glazer Peter M. , Harris Lyndsay
Published: June 2012

Background: Poly (ADP-ribose) polymerase (PARP) inhibitors have demonstrated significant anti-tumor activity not only in tumors with BRCA1 or 2 gene mutations due to synthetic lethality, but also in triple-negative breast cancers (TNBC). The triple negative group of tumors may be more sensitive to PARP inhibitors due to loss of BRCA pathway activity, although this has not been proven. Several mechanisms may affect BRCA1 protein level such as epigenetic modification, negative transcriptional regulators, as well as hypoxia. Yale investigators have demonstrated that tumor hypoxia leads to loss of critical DNA repair activities, including BRCA1, RAD-51 and γ-H2AX (Bindra RS, et al. Cancer Res 2005;65(24):11597-604). We propose BRCA1 nuclear protein is the determinant of response to PARP inhibitors in sporadic breast cancer, a phenotype we will term ‘BRCA-like’. This study was designed to explore the relationship of BRCA1 loss and hypoxia in human breast tumors.

Methods: Yale archival materials consisting of 660 human breast cancer specimens were used to establish a tissue microarray (TMA). Automated QUantitative Analysis (AQUA) was used to detect the intensity of BRCA1 and CAIXwithin a specific subcellular compartment. Carbonic Anhydrase IX (CA-IX), a downstream target of hypoxia-induced factor 1a (HIF-1a), is an accepted surrogate biomarker for tumor hypoxia. CA-IX antibody M75 was provided by the J Zavada lab (1:10000), BRCA1 monoclonal antibody M110 (Ab-1) was purchased from Calbiochem (1:1000).

Results: In the cohort of 660 specimens, 22 were found to have high CA-IX expression score (above cut-off value of 10). Cut-off was based on known positive cell lines and corresponding AQUA scores, as well as visual confirmation of positivity. A negative correlation (Rho=−0.6, p=0.0165) of BRCA1 nuclear protein with CA-IX level was found in hypoxic tumors. Of note, 14 out of 22 hypoxic breast tumors were from triple negative breast cancers (TNBC) (p=0.0034). Total number of TNBC in this cohort is 112 out of 660. BRCA1 nuclear expression alone did not correlate with overall survival (p=0.76). Conclusions: In human breast tumors BRCA1 nuclear expression is negatively correlated with tumor hypoxia, measured by CAIX. Ongoing clinical trial will determine if tumors with high CA-IX or low BRCA1 benefit from PARP inhibitor therapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C21.
Abstract C240: Protein photoadducts of acridine photosensitizers
Authors: Snapka Robert M. , Salama Samir A.
Published: June 2012

Photodynamic therapy is used to treat many cancers and some non-cancerous conditions. It exploits photosensitizers, compounds that are only toxic to cells when light exposure causes them to generate reactive oxygen species that damage many cellular molecules and organelles. A common observation is the shrinkage of distant unirradiated tumors as the result of photodynamic treatment of a single tumor. This systemic response against tumors is thought to be immunological and to involve inflammation and cytokines generated by the treated tumor in response to photodynamic damage. The possible role of photodynamic damage to proteins in such a global immune response has been underexplored. We hypothesized that photodynamically damaged tumor cell proteins might act as immunological adjuvants, stimulating an immune response to tumor specific proteins so that the photodynamically treated tumor acts as a tumor vaccine. Photoaddition of the photosensitizing molecule to cellular proteins could be one molecular mechanism for this since such adducts would be expected to produce highly immunogenic haptens on tumor cell proteins. In a test of this hypothesis, we have investigated photoaddition of acridines to cellular proteins. Cultured mammalian cells were treated with radiolabeled proflavine or acriflavine and light. SDS gel electrophoresis and gel fluorography revealed that a wide range of cellular proteins were radiolabeled. Proteinase treatment of samples before SDS PAGE resulted in label migrating with the dye front. Proteins from cells treated with the acridines but not exposed to light were not radiolabeled. Cesium chloride ultracentrifugation of cell lysates also confirmed that the only macromolecules labeled were proteins. The singlet oxygen quencher histidine and the singlet oxygen stabilizer deuterium oxide had little effect on photolabeling of cellular proteins, suggesting that the mechanism does not rely on singlet oxygen. Experiments with purified proteins and fluorography of SDS polyacrylamide gels demonstrated photoadducts of proflavine and acriflavine to these proteins. Finally, proflavine was shown to form photoadducts to angiotensin by proteomic analysis. The detailed photochemistry and the identity of the amino acid residues involved in photoadduct formation are currently being studied. We conclude that photodynamic treatment of cells can result in photoaddition of the photosensitizing drug to cellular proteins and can generate potentially immunogenic haptens.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C240.
Abstract C259: pVHL function is involved in cell death induced by YC-1 in renal cell carcinoma
Authors: Unno Yuka , Oikawa Tsuyoshi , Sawada Jun-ichi , Asai Akira
Published: June 2012

Renal cell carcinoma (RCC) is quite resistant to chemotherapy, whereas immunotherapeutic agents, including IL-2 and IFN-α, are only modestly effective with response rates of 10–20%. Novel and more effective therapeutic agents are aspired to alter the natural history of advanced RCCs. Since the protein level of von Hippel-Lindau tumor suppressor (pVHL) was extremely low in 75% of RCCs, importance of small molecules that selectively target pVHL-deficient RCC has been highlighted recently. In this study, we evaluated several known anti-cancer agents to find small molecules that induce growth inhibition according to pVHL level in RCC cells.

Our screening efforts identified YC-1, known as an anti-cancer agent that has two distinct biological actions, elevation of intracellular cGMP and inhibition of transcription factor HIF-α. In RCC cell lines we examined, YC-1 showed more effective growth inhibition to pVHL-positive RCC cells as compared to RCC cells expressing reduced level of pVHL. Cell cycle analyses revealed that YC-1 caused delay of S-phase progression and subsequent induction of apoptosis more effectively in pVHL-positive RCC cells. Under the treatment of YC-1, phosphorylation of S-phase checkpoint proteins ATM and CHK2 were elevated dramatically, and cleavages of caspase-3/9 and PARP-1, molecular hallmarks of cells undergoing apoptotic events, were promoted. However, no obvious DNA damage was observed when the cells were exposed to YC-1 at the sufficiently high concentration.

This YC-1-induced preferential apoptotic cell death in pVHL-positive RCC cells did not result from its known effects, assessed by sensitivity to the other soluble guanylate cyclase activators and by HIF-1α level. No difference was observed in growth rates of RCC cells in spite of pVHL level when the cells were exposed to BAY41-2272, a YC-1 analog, at the concentration enough to up-regulate intracellular cGMP level. In addition, as pVHL plays a key role in down-regulation of HIF-1α protein, pVHL-deficient RCC cells usually showed less sensitivity to YC-1, even though they contain remarkably higher expression of HIF-1α.

Our findings demonstrated that VHL expression sensitizes RCC cells to YC-1. Taken together with the previous report that pVHL functions as an inhibitor to RCC cell apoptosis induced by cellular stress, the results imply that YC-1 targets the pVHL-dependent survival pathway by unknown mechanisms. Thus, YC-1 is considered as a new lead compound of anticancer agents for target therapy of pVHL-positive RCCs.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C259.
Abstract C40: Phase 0 cancer trials: Ethical and regulatory issues
Authors: Silvia Camporesi
Published: June 2012

The aim of my PhD project is the analysis of the ethical and regulatory issues of Phase 0 oncological trials. In 2003, the European Agency for the Evaluation of Medicinal Products (EMEA) published a concept note followed by a Position Paper on the nonclinical safety studies needed to support human clinical trials with a single microdose of a pharmacologically active compound. In 2004, the American FDA published a draft guidance regulating early human screening studies, and in 2006 new industry guidelines for early exploratory drug studies in humans have been issued. After the publication of the US FDA guidelines, the NCI in Bethesda started the' Phase 0 program' and launched the first Phase 0 trial.

Given the recent development of such trials, there are few empirical data available on which to evaluate the ethical concerns they raise. I will pursue my analysis on the data of the NCI Phase 0 program, and by performing a comparison with the ethical concerns raised by other early cancer trials. I will also compare FDA to EMEA guidelines.

The ‘therapeutic misconception’, which is a widespread ethical problem for participants to Phase 1 cancer trials, should not be an issue of Phase 0 trials, as they declare their lack of therapeutic intent. Another ethical issue appears controversial, though, as in Phase 0 studies the interests of society seem to play a greater role than the interests of the subjects, who contribute to the progression of medical knowledge, without receiving any direct benefit from their participation. Some ethicists suggest that paragraph # 6 of Helsinki Declaration, according to which the ‘interests’ of human subjects must always take precedence over the interests of science and society, is being violated. It is time to reflect upon the nature of statements contained in Helsinki Declaration, as recently scholars such as the British bioethicist John Harris and the US Rosamund Rhodes have argued. I will discuss how the concept of ‘interest’ in participating to research should be understood, and contribute to the debate about who should be entitled to decide regarding participation to clinical trials.

On the basis of my preliminary analysis, I will conclude that Phase 0 trials appear ethically less problematic than Phase 1 cancer trials. I will also argue that the widespread presumption, that participation to clinical research is super-erogatory, needs to be changed, in favor of an approach with considers participation to research within the broadly conceived interests of the participants.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C40.
Abstract C41: Phase II pilot study of induction erlotinib therapy in patients with resectable non-small cell lung cancer to select postoperative adjuvant therapy
Authors: Lee Dae Ho , Hong Yong Sang , Kim Tae Won , Jung Kyung Hae , Kim Sang-We , Suh Cheolwon , Lee Jung-Shin , Kim Heung Tae
Published: June 2012

Background: Adjuvant chemotherapy has been widely accepted as the standard therapy for stage I-IIIA NSCLC, but resulted in significant toxicity and short-term deterioration of quality of life. In addition, not all patients can receive cytotoxic adjuvant chemotherapy. On the other hand, erlotinib demonstrated to significantly improve survival outcome for previously treated NSCLC patients regardless of their clinical characteristics. Based on those findings, we conducted a prospective pilot study of short-term induction erlotinib to select responders who might benefit from adjuvant erlotinib therapy in patients who were scheduled to undergo surgical resection.

Methods: Patients had previously untreated, potentially surgical resectable, stage IB-IIIA NSCLC with ECOG PS of 0–1 and adequate organ functions. Induction erlotinib 150 mg once a day was administered for 28 days. Subsequently patients underwent restaging and planned surgery. Erlotinib responders, defined as their tumor shrinkage of more than 10%, would receive adjuvant erlotinib for 12 weeks while the others would receive any kind of adjuvant platinum doublets. Postoperative radiotherapy could be given in case of incomplete resection or clinician's decision.

Results: Between October 2006 and March 2009, 16 patients participated in this study; median age was 59 (41–71); 13 were male; 6 were never-smoker; and 13 had adenocarcinoma. Preoperative clinical stage was as follows; IB 4, IIA 1, IIB 5, IIIA 6. All patients received induction erlotinib monotherapy and all but one, who declined surgery but received curative radiotherapy, underwent planned surgical resection. Out of 16 patients, 5 patients (3 PRs and 2 SDs) received adjuvant erlotinib therapy, while an adjuvant platinum doublet was given in 6, concurrent chemoradiation in 3 due to incomplete resection in 3 and no adjuvant therapy in 1 due to his pathologic stage of IA. There was no complete response. With a median follow-up of 24 months, 8 patients progressed and 3 patient died of progressive disease. No relationships were observed between the responsiveness and biomarkers, such expression levels of VEGF, EGFR, Ki67 and P53. Additional biomarker studies are now ongoing.

Conclusions: Induction erlotinib is tolerable and modestly active in patients with resectable NSCLC. The role of adjuvant erlotinib is still not defined yet, but short-term erlotinib before curative surgical resection could predict patients who might benefit from adjuvant erlotinib therapy and avoid toxic adjuvant chemotherapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C41.