bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1616: The anti-tumor effect of TAE226, dual inhibitor of FAK and IGF-IR, on non-small-cell lung cancer with EGFR mutation
Authors: Otani Hiroki , Toyooka Shinichi , Takaoka Munenori , Sakaguchi Masakiyo , Ueno Tsuyoshi , Yamamoto Hiromasa , Soh Junichi , Kubo Takafumi , Asano Hiroaki , Tsukuda Kazunori , Yamane Masaomi , Oto Takahiro , Kiura Katsuyuki , Huh Nam-ho , Naomoto Yoshio , Miyoshi Shinichiro
Published: February 2011

Background: TAE226 is the small molecule inhibitor that has been originally considered to inhibit focal adhesion kinase (FAK) and insulin-like growth factor-I receptor (IGF-IR). FAK and IGF-IR have been reported to be up-regulated in non-small cell lung cancer (NSCLC), suggesting their important roles on pathogenesis of NSCLC. In this study, we examined the anti-tumor effect of TAE226 on NSCLC cells in vitro and in vivo. Methods: We used NSCLC cell lines consisting of 4 EGFR-mutant cell lines (PC9, H3255, HCC827, H1975) and 3 EGFR-wild type cell lines (H1819, H1299, A549). We also used PC9-derived gefitinib-resistant cell line (RPC9). Anti-proliferative effect of TAE226 was examined with MTS assay. The status of EGFR and its downstream molecules was analyzed by Western blotting. The binding affinity of TAE226 to EGFR protein in the presence of ATP was compared between mutant and wild type EGFR proteins. The effect of TAE226 on xenograft mouse models was examined. Results: The value of IC50 (µmol/L) for EGFR-mutant cells (PC-9, H3255, HCC827, H1975 [T790M], and RPC-9 [T790M]) ranged from 0.086 to 0.31 and that of EGFR wild type cells (H1819, H1299, and A549) ranged from 1.4 to 4.7, indicating that EGFR-mutant cells are sensitive to TAE226. Western blotting assay showed that TAE226 preferentially inhibited phospho-EGFR and phospho-AKT. The binding affinity of TAE226 to mutant EGFR protein was 1000 times higher compared with wild-type EGFR protein. TAE226 via oral administration with dose of 30 mg/kg and 60 mg/kg exhibited significant inhibition of tumor growth in EGFR-mutant tumors compared with EGFR-wild type tumors without major toxicity or weight loss. These results suggested that safety of TAE226 within the treatment dose for EGFR-mutant cells in vivo. Conclusions: We indicated that TAE226 showed anti-tumor effect on EGFR-mutant cells and overcame resistance to gefitinib. Our results suggest that TAE226 will be expected as a novel strategy for NSCLC with EGFR mutation regardless of the presence of secondary T790M mutation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1616.
Abstract 1623: The nucleotide metabolizing enzyme dUTPase confers resistance to thymidylate synthase inhibition in non-small cell lung cancer cells
Authors: Wilson Peter M. , LaBonte Melissa J. , Ladner Robert D.
Published: February 2011

Chemotherapeutic agents which target the enzyme thymidylate synthase (TS) have demonstrated efficacy and seen wide-spread clinical implementation in a variety of tumor types for many decades. However, despite significant research efforts to date, little is known about the molecular pathways that regulate clinical response and resistance to TS-inhibiting agents including the fluoropyrimidines. Lung cancer is the major cause of cancer-related deaths worldwide and non-small cell lung cancer (NSCLC) is the most frequent sub-type. Several chemotherapeutic agents that target TS are currently employed in NSCLC treatment including capecitabine, S-1 and pemetrexed. Increased expression of TS is associated with resistance to TS-directed agents in a variety of tumor types including NSCLC. In this study, we analyzed the response of 4 NSCLC cell lines to TS-inhibition. All NSCLC cell lines analyzed demonstrated marked resistance to fluoropyrimidine treatment in the MTS growth inhibition assay despite the presence of TS-inhibited ternary complex. Subsequent analysis revealed that TS gene promoter activity, mRNA and protein expression is induced in NSCLC cell lines exposed to clinically relevant concentrations of 5-FU, FUdR and pemetrexed providing a rational explanation for the resistant phenotype. However, depletion of TS by RNA interference (RNAi) had no sensitizing effects to treatment with the specific TS inhibitor FUdR, suggesting that the induction of TS is not the major determinant of resistance. Conversely, depletion of the nucleotide metabolizing enzyme dUTPase by RNAi completely reversed resistance to FUdR in the MTS assay and potently inhibited colony formation in all 4 NSCLC cell lines analyzed. dUTPase depletion in combination with FUdR treatment was associated with increases in cytotoxic dUTP pools, DNA damage and apoptotic cell death. The results of this study indicate that depletion of dUTPase can rapidly sensitize NSCLC cells to otherwise non-lethal doses of a TS inhibitor. The clinical utility of a dUTPase inhibitor may have the potential to significantly increase antitumor activity in NSCLC patients treated with TS-directed agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1623.
Abstract 1627: Requirement for the Src family kinase, Fyn, in proliferation of BCR-ABL1 transduced bone marrow and aneuploidy in CML
Authors: Lu Xiaolin , Howard Adrienne , Gao Yin , Singh Melissa , Chandra Joya
Published: February 2011

Oncogenic signals stemming from the BCR/ABL kinase drive the development and progression of chronic myeloid leukemia (CML). Hence, therapies blocking kinase activity have proven extremely successful at managing this hematological malignancy. However, treatment of the terminal and progressed state of CML, blast crisis, remains difficult. Previous work from our group has identified the Src family kinase, Fyn, as being upregulated in blast crisis CML. We also reported that knockdown of Fyn slowed CML growth in vitro and in vivo and sensitized cells to BCR-ABL1 kinase inhibition with imatinib. Recent work using microarray profiling of kinase inhibitor resistant cells from a separate group has shown that imatinib resistance is associated with Fyn expression. In the current study, we utilized bone marrow cells from Fyn deficient mice to test the requirement for Fyn in p210 BCR-ABL1 mediated survival. Bone marrow cells from Fyn knockout and Fyn wildtype mice were harvested and transduced with a GFP expressing retroviral construct (MigR1) or MigR1-p210 BCR-ABL1. Cells were sorted for GFP expression and BCR-ABL1 expression was confirmed by Western blotting. Cell growth in culture was monitored over the course of three weeks in Fyn deficient and wildtype cells expressing BCR-ABL1. Fourteen days post sorting, the number of cells lacking Fyn were more than 50% lower than Fyn wildtype cells transduced with BCR-ABL1. Clonogenic assays further indicated that Fyn significantly promotes BCR-ABL1 induced bone marrow cell growth. Colony numbers in Fyn deficient cells expressing BCR-ABL1 were less than half that seen in Fyn wildtype cells. To address the role for Fyn kinase activity in these effects, constitutively active and dominant negative Fyn constructs have been transduced into BCR-ABL1 expressing cells. Constitutively active Fyn caused a high degree of chromosomal aberrations, fusions and aneuploidy suggestive of genomic instability. Taken together, these data highlight a significant role for Fyn in BCR-ABL1 mediated proliferation and secondary chromosomal aberrations, which are both features characteristic of blast crisis CML. Our results indicate that strategies to inhibit Fyn expression and kinase activity will be therapeutically relevant for progressed and/or refractory CML patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1627.
Abstract 1637: The MEK/Erk pathway is required for PC3 cell survival following FdUMP[10] treatment
Authors: Gomes Evan , Jennings-Gee Jamie , Gmeiner William H.
Published: February 2011

We have demonstrated that the novel polymeric fluoropyrimidine (FP) antitumor agent FdUMP[10] is efficacious towards prostate tumor (PC3) xenografts at well-tolerated doses while 5-fluorouracil (5FU) is not efficacious. FdUMP[10] treatment of PC3 cells results in complete inhibition of thymidylate synthase and S-phase arrest with generation of extensive DNA double-strand breaks as detected from γH2AX phosphorylation. S-phase arrest was confirmed by Western blots evaluating cyclin expression. In addition, we show that although cell-cycle arrested, FdUMP[10]-treated PC3 cells are not quiescent as indicated by extensive levels of DNA synthesis. The elevated DNA synthesis levels are likely indicative of futile repair. Although approximately 90% of cells are non-viable as assessed by a clonogenic assay, only about 30% of cells undergo apoptosis as assessed by Annexin V+ cells. The observation that FdUMP[10] treatment resulted in potent anti-tumor activity with minimal induction of apoptotic events lead us to assess to what extent activation of cell survival pathways inhibited cell death. The phosphoinositide 3-kinase PI3K/Akt and the mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinases (MEK/ERK1/2) signal transduction pathways play critical roles in mammalian cell survival and have been shown to be activated in various cancers. Previous studies have shown that inhibition of PI3K/Akt or MEK/ERK1/2 can each enhance the effectiveness of cytotoxic chemotherapy. Our results show that FdUMP[10] treatment reduced phosphorylated Akt levels suggesting FdUMP[10] treatment inactivates the PI3K/Akt pro-survival pathway. However, inhibition of PI3K/Akt with LY294002 alone promoted a significant loss in the clonogenic capacity of PC3 cells independent of FdUMP[10] treatment while co-treatment did not provide an enhanced therapeutic effect. In contrast to these findings, inhibition of the MEK/ERK1/2 pathway with U0126 showed a significant decrease in viability only in combination with FdUMP[10] treatment. Here, we demonstrate that the potent effects of FdUMP[10]-induced cytotoxicity towards prostate cancer cells may be exacerbated by impairment of the MEK/ERK1/2 but not the PI3K/Akt cell survival pathway. Altogether, our studies suggest advanced prostate cancer may be amenable to treatment with FdUMP[10] in combination with inhibitors of MAPK signaling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1637.
Abstract 1644: Sunitinib primarily acts on tumor endothelium but not tumor cells to inhibit renal cell carcinoma tumor growth
Authors: Huang Dan , Ding Yan , Li Yan , Luo Wang-Mei , Zhang Zhong-Fa , Snider John , VandenBeldt Kristin , Qian Chao-Nan , Teh Bin Tean
Published: February 2011

Sunitinib, a small-molecule inhibitor of multiple receptor tyrosine kinases (RTKs), is considered the standard of care for first line therapy of advanced clear cell renal cell carcinoma (ccRCC). However, a complete understanding of its targets and mechanism of action in the treatment of ccRCC remains incomplete. We aimed to evaluate the primary targets of sunitinib in the treatment of ccRCC (i.e. tumor cells versus endothelial cells) and to determine which RTK(s) specifically contribute to the therapeutic effect of sunitinib. Microarray gene expression profiling of human ccRCC samples showed that, of the known RTK targets of sunitinib, only PDGFR-β and VEGFR-2 were overexpressed in ccRCC relative to normal tissues. Western blotting of ccRCC and endothelial cell lines confirmed that PDGFR and VEGFR, but not other sunitinib targets, were expressed in these cells. In vitro studies found that sunitinib was unable to inhibit survival or proliferation of ccRCC cells at pharmacologically relevant concentrations (∼0.1 μM) which inhibit RTK targets. In contrast, sunitinib inhibited endothelial cell proliferation and invasion at these concentrations through suppression of VEGFR-2 signaling. Sunitinib inhibited growth of ccRCC xenografts and decreased tumor microvessel density as soon as 12 hours post-treatment; however, sunitinib showed no significant effects on tumor cell proliferation or apoptosis after up to 72 hours post-treatment. Our studies indicate that sunitinib inhibits ccRCC growth primarily through an anti-angiogenic mechanism and not through direct targeting of ccRCC tumor cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1644.
Abstract 1645: The phosphorylation status of BAD pathway influence to chemo-sensitivity
Authors: Chon Hye Sook , Marchion Douglas C. , Xiong Yin , Chen Ning , Bicaku Elona , Apte Sachin , Wenham Robert , Lancaster Johnathan M.
Published: February 2011

Objective: Patients with advanced or recurrent endometrial cancer have a poor prognosis in part due to lack of efficacy of currently available chemotherapies and incomplete understanding of the biology that underlies chemo-response. In the current study we used genomic expression data to identify molecular signaling pathways associated with endometrial cancer cell resistance to cisplatin, doxorubicin chemotherapy and further characterized the role of the BAD apoptosis pathway as a therapeutic target and determinant of chemo-response.

Methods: Eight endometrial cell lines were subject to genome-wide expression analysis using Affymetrix HG-U133Plus GeneChips and in parallel, ranked by relative sensitivity to cisplatin and doxorubicin using MTS cell proliferation assays. Gene array data from sensitive versus resistant cell lines was compared by student's t-test and Pearson's correlation to identify those genes associated with drug resistance and further evaluated by GeneGo Metacore software for representation of molecular signaling pathways. Furthermore, the role of the BAD pathway in chemo-resistance was evaluated by selective inhibition of BAD pathway members, PKA, PP2C, AKT1, AKT2 and AKT3 by siRNA or pharmacologic agents in the presence and absence of cisplatin. Cell death and growth arrest in the presence of cisplatin were evaluated by Western blot for PARP cleavage as well as nuclear condensation and fragmentation and MTS assays.

Results: Student's t-test and Pearson's correlation identified the expression of 325 and 1136 genes to be associated with cisplatin (p<0.01) and doxorubicin (p<0.01) resistance, respectively. Pathway analysis of genes associated with both cisplatin and doxorubicin resistance indicated significant representation of the BAD apoptosis pathway (p<0.002). Selective depletion of the phosphatase, PP2C by siRNA resulted in increased phosphorylation of BAD, serine 118 and increased resistance to cisplatin induced cell growth arrest and cell death. In contrast, depletion of the kinase, PKA resulted in decreased phosphorylation of BAD, serine 118 and increased cell death in the presence of cisplatin. In addition, depletion of AKT1 using siRNA as wells as treatment of cells with the AKT inhibitor, triciribine, increased cisplatin-induced growth arrest and cell death, whereas depletion of AKT2 or AKT3 did not affect cisplatin sensitivity.

Conclusions: The phosphorylation status BAD may influence endometrial cancer cell sensitivity to chemotherapy and represents a compelling target for future therapeutic development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1645.
Abstract 1647: Imatinib mesylate leads to modulation of regulators of tumor cell quiescence in gastrointestinal stromal tumors (GIST) cells
Authors: Seneviratne Danushka S. , Parry Joshua A. , Baron Julianne L. , Duensing Stefan M. , Duensing Anette U.
Published: February 2011

Gastrointestinal stromal tumors (GISTs) are caused by activating mutations in the KIT or PDGFRA receptor tyrosine kinase genes. While GISTs can successfully be treated with imatinib mesylate, many patients retain detectable tumor mass during treatment and experience relapse following termination of treatment. We wanted to determine whether these clinical observations result from direct, imatinib-induced effects on GIST cells. We have shown that imatinib can directly induce tumor cell quiescence in a proportion of GIST cells through an increase of nuclear CDH1, an activator of the anaphase-promoting complex (APC). This event was associated with downregulation of the F-box protein SKP2 and subsequent upregulation of its target, the CDK inhibitor p27(Kip1). Exit from the cell division cycle was reflected by reduced BrdU incorporation and reduced cyclin A protein expression. Quiescent tumor cells were able to re-enter the cell division cycle five days after removal of imatinib as indicated by enhanced BrdU incorporation, decrease in p27(Kip1) expression and increase of cyclin A expression. Furthermore, high SKP2 and low p27(Kip1) levels correlated with an increased risk of progression. Mechanistically, imatinib was found to modulate components of the human DREAM complex, a key regulator of quiescence. Imatinib treatment resulted in upregulation of p130 and downregulation of E2F4 as well as LIN37 and LIN9. After removal of imatinib, levels of p130 decreased, whereas E2F4, LIN37 and LIN9 levels increased, as a reflection of the cells re-entering the cell cycle. Our results indicate that GIST cells that do not undergo apoptosis in response to imatinib enter quiescence and are hence temporarily removed from the proliferative pool However, cells were found to readily re-enter the cell division cycle in the absence imatinib. Our results encourage further studies to target the quiescence pathway in GISTs therapeutically.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1647.
Abstract 1648: SCH 1473759, a novel aurora inhibitor, demonstrates enhanced antitumor activity in combination with taxanes and KSP inhibitors
Authors: Basso Andrea D. , Gray Kimberly , Tevar Seema , Ponery Samad , Lee Suining , Liu Ming , Smith Elizabeth , Yu Tao , Tagat Jay , Doll Ronald , Belanger David , Siddiqui Arshad , Zhang Yonglain , Xiao Yushi , Esposite Sara , Monsma Fredrick , Hicklin Dan , Kirschmeier Paul
Published: February 2011

Aurora kinases are required for orderly progression of cells through mitosis. Inhibition of these kinases by siRNA or a small molecule inhibitors results in aberrant endoreduplication and cell death. SCH 1473759 is a novel Aurora inhibitor with potent mechanism based cell activity. The compound is active against a large panel of tumor cell lines from different tissue origin and genetic backgrounds. We found that asynchronous cells require 24 hour exposure to SCH 1473759 to induce maximal endoreduplication and cell kill. However, following a taxane or KSP inhibitor mitotic arrest, less than 4-hour exposure was sufficient to induce endoreduplication. This finding correlated with the ability of SCH 1473759 to accelerate exit from mitosis in response to taxane and KSP induced arrest, but not that of a nocodazole arrest. SCH 1473759 demonstrated single agent biomarker and anti-tumor activity in A2780 ovarian xenograft models. Further, efficacy was enhanced in combination with taxotere and found to be most efficacious when SCH 1473759 was dosed 12-hours post taxotere. These findings could have clinical implications for the development of Aurora inhibitors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1648.
Abstract 1654: Compensatory insulin receptor (IR) activation upon inhibition of insulin-like growth factor receptor (IGF-1R): Rationale for co-targeting IGF-1R and IR in cancer
Authors: Buck Elizabeth , Gokhale Prafulla , Koujak Susan , Eyzaguirre Alexandra , Wild Robert , Epstein David , Pachter Jonathan , Miglarese Mark
Published: February 2011

The insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase (RTK) and a critical mediator of signaling through the PI3K-AKT pathway. IGF-1R is required for oncogenic transformation and tumorigenesis, and inhibiton of IGF-1R results in reduced proliferation and survival of tumor cells. These observations have spurred intense drug discovery and development efforts for both biologic and small molecule IGF-1R inhibitors for the treatment of cancer.

The ability for one RTK to compensate for another to maintain growth and survival signaling in tumor cells is emerging as a common mechanism of resistance to anti-tumor agents that selectively target individual RTKs. As IGF-1R is structurally and functionally related to the insulin receptor (IR), and IR can also activate tumor cell AKT signaling and cellular transformation, we asked whether IR signaling can contribute to resistance to IGF-1R inhibition in tumor cells.

In a panel of human tumor cell lines, IGF-1R/IR crosstalk was observed after treatment with a selective anti-IGF-1R monoclonal antibody, MAB391. Tumor cells treated with MAB391 responded with a compensatory increase in phospho-IR, which was also associated with an inability to fully inhibit phospho-IRS1 and phospho-AKT. In contrast, treatment with OSI-906, a small molecule dual kinase inhibitor of IGF-1R and IR, resulted in enhanced inhibition of the IRS1-AKT signaling pathway. OSI-906 showed superior efficacy compared to MAB391 in human tumor xenograft models where both phospho-IR and phospho-IGF-1R were detectable, and presumably both receptors were required by tumor cells for growth and/or survival. Both insulin and IGF-2 can activate the IR-AKT pathway and we show that treatment with either growth factor resulted in decreased sensitivity of tumor cells to MAB391, but not OSI-906. In tumor cells with an autocrine IGF-2 signaling loop, both OSI-906 and an anti-IGF-2 neutralizing antibody reduced phospho-IR and phospho-AKT levels, whereas MAB391 was ineffective. Collectively, these data indicate that OSI-906, which co-targets IGF-1R and IR, may provide superior efficacy as compared to agents that selectively target only IGF-1R.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1654.
Abstract 1658: Fenretinide induces lethal autophagy in pancreatic cancer cells
Authors: Messner Maria C. , Cabot Myles C.
Published: February 2011

Background: Recent studies show that the synthetic vitamin A analog fenretinide (4-HPR) is cytotoxic in a number of human tumor cell lines and is a chemopreventative agent that is well-tolerated clinically. 4-HPR has been shown to augment intracellular levels of reactive oxygen species (ROS) and ceramides, and to activate the MAPK family of kinases. Pancreatic cancer cells are sensitive to increased levels of ROS, ceramides, and MAPK phosphorylation, suggesting 4-HPR will be an effective agent against this disease.

Results: 4-HPR exposure markedly decreased viability of MIA PaCa-2 and PANC-1 cells, as shown by a dose-dependent decrease in absorbance from an MTS assay and a 3-fold increase in propidium iodide staining. A 50% reduction in [3H]thymidine incorporation showed that cell proliferation was halted as well. 4-HPR treatment caused a 3-fold increase in de novo ceramide production at 24 h and a time- and dose-dependent generation of ROS. 4-HPR led to a minor increase in caspase 3/7 activity and in FITC-Annexin V-positive cells. However, other measures of apoptosis were lacking, suggesting apoptosis was not the major death pathway elicited by 4-HPR. Further investigation of the mechanism of cell death suggests that the dominant pathway triggered by 4-HPR is lethal autophagy. After 4-HPR treatment, microscopic analysis of cells exposed to acridine orange showed bright red autophagic vesicles, and Western blot showed a robust increase in LC3 II protein expression. Pretreatment with the autophagy inhibitor 3-methyladenine (3-MA) or the ROS scavenger vitamin E (VE) abrogated the increase in LC3 II expression caused by 4-HPR exposure, and both 3-MA and VE partially preserved viability. A sustained, dose-dependent increase in phosphorylation of the stress kinases JNK and p38 was observed following incubation with 4-HPR, whereas phosphorylation of the mitogenic kinase ERK 1/2 decreased. Pretreatment with myriocin (an inhibitor of de novo ceramide production), 3-MA, or VE attenuated the increase in JNK and p38 phosphorylation caused by 4-HPR. Additionally, VE pretreatment blocked the decrease in ERK 1/2 phosphorylation elicited by 4-HPR.

Conclusions: These data suggest that 4-HPR induces lethal autophagy in human pancreatic cancer cells by a mechanism that involves generation of ROS and engagement of the MAPK and sphingolipid signaling pathways. 4-HPR used alone or in combination with current therapies or signaling pathway modulators may be a promising avenue for future pancreatic cancer therapies. [Support: NIH/NIGMS grant no. GM077391]

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1658.
Abstract 1666: Regorafenib (BAY 73-4506): preclinical pharmacology and clinical identification and quantification of its major metabolites
Authors: Zopf Dieter , Heinig Roland , Thierauch Karl-Heinz , Hirth-Dietrich Claudia , Hafner Frank-Thorsten , Christensen Olaf , Lin Tiffany , Wilhelm Scott , Radtke Martin
Published: February 2011

Regorafenib is a novel diphenylurea oral multikinase inhibitor of angiogenic, stromal, and oncogenic kinases with potent preclinical antitumor activity and long-lasting antiangiogenic activity (as measured by dynamic contrast-enhanced magnetic resonance imaging).

Regorafenib treatment has recently shown to result in a 31% partial response (PR) rate and 50% stabilization rate in patients with metastatic renal cell carcinoma in a Phase II study. Promising clinical activity was shown in a Phase I study in 27 evaluable patients with advanced refractory colorectal cancer (median 4 prior regimens) with a 74% overall disease control rate (PR + stable disease).

The biotransformation of regorafenib was investigated in vitro with liver microsomes and hepatocytes, and in vivo in the plasma of several species. In man, the N-oxide (M-2) and the demethylated N-oxide (M-5) appear significant as both metabolites show systemic exposure (area under the curve [AUC], mg*h/L) at steady state similar to regorafenib (the parent compound) in patients at a dose of 160 mg in a 3 weeks on/1 week off Phase I study.

Characterization of both M-2 and M-5 demonstrated potent pharmacologic activities. In biochemical kinase assays, M-2 and M-5 showed an inhibition profile similar to but distinct from regorafenib. In cellular assays, M-2 and M-5 inhibited key targets such as vascular endothelial growth factor (VEGF) receptor 2, TIE-2, and mutant and wild-type c-KIT and B-RAF, with IC50 values very similar to regorafenib for M-2 and somewhat higher for M-5.

Regorafenib and both M-2 and M-5 dosed iv at 1 mg/kg showed significant activity in a rat VEGF hypotensive pharmacodynamic model. Both metabolites dosed orally exhibited potent dose-dependent tumor growth inhibition (TGI) in preclinical murine HT-29 colorectal and MDA-MB-231 breast cancer xenografts, achieving significant TGI of 62/58% and 54/50%, respectively, compared with vehicle controls at 10 mg/kg. Regorafenib revealed 66-77% TGI at 10 mg/kg in these models. On oral administration of regorafenib to mice, the N-oxide (M-2) plasma level accounted for ∼20% of the AUC of regorafenib; conversely, on administration of M-2, regorafenib exposure reached ∼20% of M-2 exposure, indicating that oxidation to and reduction of the N-oxide are metabolic pathways in vivo.

In summary, these data demonstrate that the metabolites M-2 and M-5, exhibiting similar exposure as regorafenib, their parent compound, in man, are pharmacologically active and therefore most likely contribute to the clinical antitumor activity of regorafenib.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1666.
Abstract 167: Identification of differentially methylated genes in normal prostate tissues from African American and Caucasian men
Authors: Kwabi-Addo Bernard , Wang Songping , Lee Norman H. , Estecio Marcos R. , Jelinek Jaroslav , Chung Woonbok , Ittmann Michael M.
Published: February 2011

BACKGROUND. Prostate cancer (PCa) is a common malignancy and a leading cause of cancer deaths among men in the United States. African American men have both a higher incidence and significantly higher mortality rates than Caucasian men. Abundant evidence has accumulated to suggest that epigenetic DNA methylation changes may appear earlier during PCa development than genetic changes, as well as more commonly and consistently. Most studies have emphasized DNA hypermethylation as an important mechanism for inactivation of key regulatory genes in prostate cancers. Thus methylated genes can serve as biomarkers for the detection of cancer from clinical specimens such as tissue biopsies or body fluids. However, very few studies have investigated differences in DNA methylation pattern in different ethnic groups or their application as ethnic sensitive biomarkers for disease detection. We hypothesize that differential methylation patterns maybe associated with ethnicity and could potentially contribute to the incidence and mortality of prostate cancer. METHODS. We have identified several genes to be differentially methylated in human prostate cancer cell line using methylated CpG island amplification coupled with CpG promoter microarray. Pyrosequencing was used to quantitatively measure the methylation levels of CDH11, FOXN4, PAX9, TIMP3, RPRM, SPARC, TCF3,CYP27B1, RARβ2, AR, GSTP1, NKX2-5, RASSF1A and TIMP3 genes in prostate cell lines, matched pairs of benign and prostate cancer tissues from individual prostate cancer patients, and normal prostate biopsies from African American and Caucasian men. Real time PCR was used to determine gene expression at the RNA transcript level. RESULTS. DNA hypermethylation was observed for NKX2-5, FOXN4, TIMP3, RPRM, TCF, SPARC, GSTP1, RASSF1A, RARβ2 and SPARC in prostate cancer cells and prostate cancer tissues samples. Higher frequency of methylation was observed for several genes including NKX2-5, GSTP1, RASSF1A, and RARβ2 genes in punch biopsies of normal prostate samples obtained from African American men in comparison to samples from Caucasian men. CONCLUSION. Our observations suggest a genome wide differential methylation patterns in prostate tissue samples obtained from African American men versus Caucasian men. Such alterations could account for some of the biological differences underlying prostate cancer disparity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 167.
Abstract 1672: The plasma and cerebrospinal fluid pharmacokinetics of sorafenib after intravenous administration in non-human primates
Authors: Kim AeRang , McCully Cindy , Cruz Rafael , Cole Diane E. , Fox Elizabeth , Balis Frank M. , Widemann Brigitte C.
Published: February 2011

Background: Sorafenib is an oral multikinase inhibitor that targets intracellular and receptor protein kinases thought to be crucial in the pathogenesis and growth of many tumors including brain tumors. Sorafenib is FDA approved for advanced renal cell and hepatocellular carcinoma. Clinical trials in primary and metastatic brain tumors are ongoing. We evaluated the plasma and cerebrospinal fluid (CSF) pharmacokinetics (PK) of sorafenib after an intravenous (IV) dose in a non-human primate (NHP) model. Methods: Sorafenib 7.3 mg free base equivalent/kg was administered as a 1h IV infusion in 20% cyclodextrin and normal saline to 3 NHP with IV catheters and Ommaya reservoirs. Serial paired plasma and CSF samples were drawn over 24h for PK analysis. Sorafenib was quantified with a validated liquid chromatography/tandem mass spectrometry assay. The lower limit of quantification for plasma and CSF was 5 ng/mL and 0.025 ng/mL, respectively. PK parameters were estimated using non-compartmental methods. CSF penetration was calculated from the AUC CSF : AUC plasma. Plasma free drug exposures (AUC) were calculated from published values for human plasma protein binding (99.5%). Results: IV administration of sorafenib was well tolerated in the NHP without evidence of clinical toxicity. The PK parameters of sorafenib in NHP for plasma and CSF are described in the table below. The mean CSF penetration of sorafenib was 0.02%. When corrected for plasma protein binding, the mean CSF penetration was 3.4%. Conclusions: Sorafenib is well tolerated in NHP and measureable in CSF after an IV dose. The CSF penetration of sorafenib is limited relative to total and free drug exposure in plasma.Plasma and CSF PK of sorafenib in NHPAnimalCmax plasma [µg/mL]Clearance [mL/min/kg]AUC 0-24h plasma [µg•h/mL]Cmax CSF [µg/mL]AUC 0-24h CSF [µg•h/mL]CSF penetration [%]17.62.231.20.000450.00420.01326.42.029.60.000580.00660.02233.41.323.20.000460.00340.015Mean5.81.728.00.000500.00480.02SD2.10.54.30.000070.00160.005

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1672.
Abstract 1673: A pharmacokinetics/pharmacodynamics evaluation of the concentration-QTc relationship of PF-02341066 (PF-1066), an ALK and c-MET/HGFR dual inhibitor administered orally to patients with advanced cancer
Authors: Nickens Dana , Tan Weiwei , Wilner Keith , Camidge D. Ross , Shapiro Geoffrey , Dezube Bruce , Clark Jeffrey
Published: February 2011

Objective: Assess the potential effect of PF-1066 plasma concentrations on the observed RR corrected QT interval in patients with advanced cancer.

Methods: Plasma concentrations of PF-1066 (total drug) and ECG data were collected in study A8081001, an open label, multi-center Phase 1 dose escalation, and safety, pharmacokinetic and exploratory study (J Clin Oncol 27:15s, 2009 (suppl; abstr 3509)). PF-1066 was administered orally on an empty stomach once (QD) or twice (BID) a day in continuous 28-day cycles with doses ranging from 50 mg QD to 300 mg BID. Time-matched, triplicate (≥ 2 minutes apart) ECGs were obtained during the screening period, at pre-dose, 6 hours (∼ Cmax), and 24 hours post-dose on Cycle 1 Day 1, and at pre-dose and 6 hours (∼ Cmax) post-dose on Cycle 1 Day 15 and Cycle 2 Day 1. Baseline ECG data was used to assess which heart rate correction method (QTcB, QTcF and a study specific correction, QTcS) was most appropriate. A linear mixed effects model was used to evaluate the relationship between plasma concentrations and the RR-interval and corrected QT-interval.

Results: A total of 62 patients were available for this analysis. The majority of patients had at least 5 triplicate measurements post dose. Fifty-three percent (n=33) were male. Plasma concentrations ranged from 0.228 ng/mL to 1040 ng/mL and the median concentration over all doses was 83.2 ng/mL. At baseline, median heart rate, QTcB- and QTcF-interval were, respectively, 80 bpm, 429 msec and 408 msec. The post-treatment median heart rate, QTcB- and QTcF-interval were, respectively, 73 bpm, 432 msec and 416 msec. The concentration-RR analysis indicates that on average heart rate decreases at approximately 2.5 bpm per 100 ng/mL increase in PF-1066 concentration. QTcS was determined as the most appropriate heart rate correction method for QT-interval. The estimated slope for concentration-QTcS-interval is 0.0076 msec/ng/mL (90% conf. limits: −0.0051, 0203), indicating a small but not statistically significant positive trend in the concentration-QTcS relationship. Based on the model, the average drug associated increase in QTcS is 2.9 msec at the mean Cmax following 250 mg BID dosing.

Conclusion: Based on the PK/PD analysis, a clinically relevant QT prolongation is not expected within the therapeutic dose range (up to 250 mg BID) of PF-1066 in patients with advanced cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1673.
Abstract 1674: The NQO1 C609T polymorphism is associated with poorer response to AC adjuvant therapy in the treatment of breast cancer
Authors: Jamieson David , Cresti Nicola , Bray Johanne , Sludden Julieann , Griffin Melanie , Farmie Eve , Verrill Mark , Boddy Alan
Published: February 2011

A non-synonymous SNP in the NAD(P)H: Quinone Oxidoreductase 1 gene has previously been implicated in the response of breast cancer patients to anthracycline containing regimens in a retrospective cohort study. NQO2 is 54% homologous to NQO1 at the primary protein structure and, despite the lack of a characterised endogenous electron donating cofactor for NQO2, the two enzymes share numerous substrates. NQO2, in contrast to NQO1, is additionally capable of reducing reactive quinone metabolites of estradiol. The NQO2 gene has a non-synonymous SNP that has previously been shown to result in a lower average NQO2 activity in cohorts of bladder and ovarian cancer patients. It is unknown if the NQO2 SNP impacts on survival in breast cancer.

Genomic DNA samples from 239 women with early breast cancer were genotyped for the NQO1 (rs1800566) and NQO2 (rs1143684) SNPs. All of the participants were recruited from Medical Oncology out-patient clinics within the Newcastle upon Tyne Hospitals NHS Foundation Trust and were treated with an AC adjuvant therapy regimen with curative intent. Median follow up was 78 months and followed a standard protocol. Genotyping was done by the Taqman RT-PCR method.

The NQO1 SNP was associated with a decreased time to progression (HR 2.8, 95%CI 1.4-5.64, p=0.003), decreased overall survival (HR 3.6, 95%CI 1.4-10, p=0.007) and a lower likelihood of having a dose delay, all indicative of NQO1 mediated sensitisation to the effects of doxorubicin. The NQO2 SNP had no impact on the cohort as a whole but was associated with a decreased overall survival (p=0.009, log-rank) and time to progression (p=0.004, log rank) in patients who were oestrogen receptor (ER) negative.

This study confirms the previous observation of NQO1 genotype influencing response to anthracyclines and indicates a potential impact of an NQO2 SNP on the prognosis of ER negative breast cancer. The mechanism by which NQO2 genotype influences breast survival remains to be elucidated.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1674.
Abstract 1686: Low dose metronomic gemcitabine has high antimetastatic efficacy in an orthotopic mouse model of pancreatic cancer
Authors: Cao Hop S. Tran , Bouvet Michael , Romney Eric , Kaushal Sharmeela , Keleman Alex , Kim Ginna , Fruehauf John , Imagawa David K. , Hoffman Robert M. , Katz Matthew H.G.
Published: February 2011

Therapies for metastasis of pancreatic cancer (PC) are ineffective. We report here the efficacy of metronomic gemcitabine with and without the tyrosine kinase inhibitor sunitinib (SU) on metastasis in an orthotopic model of PC.

Mice with highly metastatic, orthotopic PC expressing red fluorescent protein (RFP) were treated with intraperitoneal gemcitabine on a metronomic (1 mg/kg daily, MET) or with maximum tolerated dose (150 mg/kg twice weekly, MTD) schedule with or without oral SU. Primary tumor growth, metastasis, and survival were evaluated. Primary tumor growth and metastasis were quantified by fluorescence imaging.

Control mice with orthotopically-implanted MIA-PaCa-2-RFP died of local and widespread metastatic disease within 4 weeks. Gemcitabine at daily doses of 2 mg or greater led to toxicity and death within 1 month in mice without tumors but MET at 1 mg/kg daily was well tolerated. Treatment of mice with orthotopic MIA-PaCa-2-RFP with MET at 1 mg/kg daily for 2 weeks dramatically suppressed metastasis at multiple sites_an effect enhanced by SU (p<0.05). Although primary tumor growth was inhibited by MET+SU (p<0.05), it was not inhibited by either MET or SU alone. Both MET and SU had a modest effect on survival compared to control but survival was limited by extensive primary tumor growth. MET+SU had a more pronounced effect on survival (p<0.05). MTD with or without SU had the most pronounced effects on primary tumor growth and survival but its antimetastatic effect was similar to that of MET+SU (p<0.05). Antimetastatic activity approaching that of standard MTD gemcitabine was achieved with a gemcitabine dose reduced 42 times from MTD using MET and was further enhanced by sunitinib. Our results suggest the clinical potential of this well-tolerated regimen of MET against PC in the adjuvant and maintenance settings.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1686.
Abstract 169: The origin of colorectal cancer with CpG island methylator phenotype
Authors: Toyota Minoru , Yamamoto Eiichiro , Suzuki Hiromu , Kamimae Seiko , Yamano Hiro-o , Shinomura Yasuhisa , Imai Kohzoh
Published: February 2011

Colorectal cancers (CRCs) are divided into three different subclasses based on the genetic and epigenetic profiles: CpG island methylator phenotype (CIMP) 1, CIMP2 and CIMP-negative. CIMP1 has been shown to be associated with BRAF mutations and sessile serrated adenoma. CIMP2 has been shown to be associated with KRAS mutation, but precursor lesions of CIMP2 remain to be determined. Our aim of this study is to identify the origin of CRCs with CIMP by examining the promoter methylation of tumor-related genes and genome-wide methylation profiles in precursor lesions. Based on histological examination after endoscopic mucosal resection or operation, the specimens were divided into two groups: 65 serrated polyps (SPs), 109 colon adenomas (ADs) with or without high grade dysplasia. Endoscopically, subsets of SPs were composed of flat and protruded lesions, and biopsy samples were obtained from both lesions, and were divided into three groups: flat lesions only (SP1, 19 cases), flat lesions adjacent to protruded lesions (SP2, 21 cases), or protruded lesions adjacent to flat lesions (SP3, 25 cases). Mutations of BRAF were observed frequently in SPs but not in ADs (26% vs 2%), indicating that the SPs are the origin of CIMP1 characterized by BRAF mutations and microsatellite instability as suggested previously. Frequencies of KRAS mutations were highest in tubulovillous adenomas (TVs) followed by SPs, and tubular adenomas (TAs) (57% vs 43% vs 20%). High frequency of KRAS mutations in SPs and TVs prompt us to examine genome-wide methylation profiles in these tumors. By hierarchical clustering analysis of the MCAM results, SP3, SP1/SP2, and normal colon were grouped into three distinct clusters, and it is suggested that genome-wide methylation occurs during the course of progression from SP2 to SP3, and was associated with acquirement of CIMP. We further examined methylation profiles of additional SPs, ADs (TVs and TAs) with KRAS mutations. Cluster analysis of KRAS mutant SPs and ADs grouped tumors into three clusters; cluster-1 with genome-wide aberrant methylation, cluster-2 with moderate methylation, and cluster-3 with little methylation. Cluster-1 contained SP3 and majority of TVs, cluster-2 contained SP1, SP2, and TAs, and cluster-3 contained normal colon. The present study suggested that CRCs with CIMP2 are originated from two distinct subgroups; CIMP2-protruded (CIMP2-P) originated from serrated-villous protruded tumors, and CIMP2-depressed (CIMP2-D) originated from tubular-depressed pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 169.
Abstract 170: Hypermethylation of DBCCR1 gene in acute myeloid leukemia
Authors: Dai Aili , Zhao Chen , Yin C. Cameron , Chen Ling , Sun Xiaoping , Dave Sanat , Huang Xiaoyan , Zhang Yu H. , Han Xin , You M. James
Published: February 2011

DNA hypermethylation likely plays a role in the tumorigenesis and prognosis of acute myeloid leukemia (AML). To identify relevant methylated genes in AML, we have compared several expression and methylation profiles. With expression analysis, we identified that DBCCR1, TRPC6, DCC and SOX9 have decreased expression levels in the most analyzed AML cell lines. Among these candidates, DBCCR1 (deleted in bladder cancer chromosomal region candidate 1) gene at chromosome 9q33 was identified as a putative tumor suppressor gene that is frequently targeted by hypermethylation in transitional cell carcinomas of the bladder. In addition, DBCCR1 was reported frequently methylated in hematological malignancies, including diffuse large B-cell lymphoma, follicular lymphoma and mantle cell lymphoma. DBCCR1 may play an important role in the regulation of cell growth and programmed cell death. But the expression levels and methylation status of DBCCR1 in AML have not been established. In this study, we analyzed the expression pattern of DBCCR1 in AML cell lines with RT-PCR analysis. DBCCR1 mRNA expression was robust in normal bone-marrow mononucleated cells but markedly diminished in all of 9 AML cell lines. DBCCR1 was methylated in all of AML cell lines (9 of 9, 100%) and its methylation was inversely correlated with DBCCR1 mRNA expression, which was demonstrated by RT-PCR and COBRA (Combined Bisulfite Restriction Analysis) analyses. Furthermore, we also detected frequent methylation (at least 90%) of DBCCR1 in multiple primary AML samples. These findings indicate that DBCCR1 is frequently silenced by hypermethylation in AML. We are investigating the functional role of DBCCR1 in the pathogenesis of AML. In addition, the diagnostic and prognostic values of methylated DBCCR1 in AML are being pursued. In summary, putative tumor suppressor gene DBCCR1 shows significantly diminished expression in AML due to frequent hypermethylation.

* Aili Dai and Chen Zhao contributed equally to the presented work.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 170.