bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract PR-1: Acquired resistance to RAF inhibitors is mediated by splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.
Authors: Poulikakos Poulikos I.
Published: June 2012

Tumors with mutant BRAF(V600E) are dependent on BRAF/MEK/ERK (ERK signaling) for their growth. ATP-competitive RAF inhibitors suppress ERK signaling in cells with mutant BRAF, but paradoxically enhance ERK signaling in cells with wild-type BRAF. We have shown previously the mechanistic basis of this phenomenon: active RAS promotes homo- and heterodimerization of RAF. When RAF exists in dimers, drug binding to one protomer transactivates the other unbound protomer. In BRAF(V600E) tumors, RAS is not activated, RAF is predominantly monomeric and ERK signaling is inhibited by the drug. These results predict that RAF inhibitors will be effective in tumors in which BRAF is mutated. Furthermore, because RAF inhibitors do not inhibit ERK signaling in other cells, the model predicts that they would have a higher therapeutic index and greater antitumor activity than MEK inhibitors. Confirming these predictions, RAF inhibitors have shown remarkable clinical activity in patients with melanomas that harbor mutant BRAF(V600E), however, resistance invariably develops. The mechanism of action of these drugs indicates that promotion of RAF dimerization (by lesions that activate RAS, or other means) could lead to drug resistance in mutant BRAF tumors. Indeed, RAS mutation has been reported as a mechanism of resistance in a subset of patients that progressed on the RAF inhibitor PLX4032 (vemurafenib). In order to identify novel mechanisms of resistance, we generated cell lines resistant to PLX4032. We found that a subset of cells resistant to PLX4032 express a 61kd splicing variant form of BRAF(V600E) that lacks exons 4–8, a region that encompasses the RAS-binding domain (p61BRAF(V600E)). p61BRAF(V600E) exhibits enhanced dimerization as compared to full length BRAF(V600E) in cells with low levels of RAS activation. In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signaling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to PLX4032, indicating that dimerization status of BRAF(V600E) determines the effects of the drug. We detected BRAF splicing variants in tumors collected at the time of progression on PLX4032, but not in matched pre-treatment tumors, supporting the clinical relevance of this mechanism and its potential utility as a therapeutic target. These data support the model that inhibition of ERK signaling by RAF inhibitors is dependent on levels of RAS-GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PR-1.
Abstract 1300: Evaluating the relative bioavailability of a new formulation of navitoclax (ABT-263) in both cancer patients and healthy volunteers
Authors: Yang Jianning , Xiong Hao , Krivoshik Andrew , Holen Kyle , Pradhan Rajendra , Chiu Yi-Lin , Nada Adel , Tolcher Anthony , Awni Walid , Humerickhouse Rod
Published: June 2012

INTRODUCTION: Navitoclax (ABT-263) is a novel, orally bioavailable small molecule that inhibits Bcl-2 family proteins and promotes apoptosis. The current Phase 1 formulation is a lipid solution, which has a short shelf-life. A new lipid solution with improved shelf-life was developed to support Phase 2/3 clinical development. Relative bioavailability studies in patients were expected to have large variability, which would make the interpretation challenging. Meanwhile, the toxicity profile of navitoclax limited an evaluation at the recommended phase II dose in healthy volunteers. Therefore, relative bioavailability studies for the new formulation were conducted in both healthy volunteers (at doses with minimal safety risk) and cancer patients (at therapeutic doses).

METHODS: Two randomized, crossover studies were conducted to evaluate the bioavailability of the new formulation relative to that of the Phase 1 formulation. Study I was conducted in 12 healthy female subjects with non-childbearing potential at a single dose of 25 mg navitoclax. Study II was conducted in 12 cancer patients at a single dose of 250 mg navitoclax. Both studies were conducted under low-fat conditions.

RESULTS: The new lipid solution was bioequivalent to the Phase 1 formulation in healthy subjects. In patients, the point estimates of the bioavailability of the new lipid solution were 30% higher than the Phase 1 formulation; nevertheless, the point estimates were associated with wide 90% confidence intervals indicating high uncertainty in the point estimate. The percent coefficient of variation for navitoclax exposure in patients (49%) was markedly higher than that in healthy subjects (25%).

CONCLUSION: Relative bioavailability assessment in healthy volunteers had substantially lower variability compared to that in cancer patients, therefore served as the primary basis for decision making. However, results from patients provided supportive information at the therapeutic dose levels. Overall results from both studies in healthy subjects and patients provided strong evidence that the new lipid solution has comparable bioavailability as the Phase 1 formulation under low-fat conditions at therapeutic doses, and enabled a decision to select the new lipid formulation without dose modification for Phase 2/3 studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1300. doi:10.1158/1538-7445.AM2011-1300
Abstract 15: Phosphorylation defective mutant Bcl-xL knockin mice exhibit behavioral abnormalities linked with neurodevelopmental disorders
Authors: Basu Aruna , Casadesus Gemma , Grisham Tina , Chen Yinhuai , Haldar Subrata
Published: June 2012

Bcl-xL, a close homolog of Bcl2, is an important regulator of apoptosis and is overexpressed in many human cancers including neuroblastoma. Previously we have discovered that the regulation of phosphorylation of Bcl-xL on serine 62 residue by tubulin-binding anticancer drugs at mitosis may function as a checkpoint to permit apoptosis of cancer cells. However, the physiological significance of Bcl-xL phosphorylation still remains obscure. With a view to comprehending the functional relevance of Bcl-xL phosphorylation in vivo, we have generated knock-in mice expressing an unphosphorylable ser62 (equivalent to human ser62), by substitution with an alanine residue. Thus placing the phosphorylation-defective mutant of Bcl-xL into the germ line of mice creates opportunities to observe the effects of endogenous phosphorylation in the context of multicellular organisms.

Since Bcl-xL has been shown to be maintained at high level in CNS, initially a full behavioral phenotyping battery was carried out with wild type (WT) and Bcl-xLS62A(+/+) mice. Born at Mendelian ratios, the Bcl-xLS62A(+/+) mice demonstrated reduced startle response and reduced PPI (Prepulse inhibition) at lower PPI amplitudes compared to WT mice. Moreover, cognitive deficits were noted in the passive avoidance task between Bcl-xLS62A(+/+) mice and WT littermates. These data indicate that mutation in the phosphorylation site of Bcl-xL drive a range of functional abnormalities and many of the observed behavioral abnormalities in Bcl-xLS62A(+/+) mice are consistent with neurodevelopmental disorders. In all, our findings suggest that endogenous phosphorylation of antiapoptotic protein Bcl-xL might play a critical role in neurodevelopment and subsequent CNS functional output. Further molecular analysis on the effect of blocking of endogenous phosphorylation of Bcl-xL on neuronal apoptosis will be discussed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 15. doi:10.1158/1538-7445.AM2011-15
Abstract 1643: AZD4547, a potent and selective inhibitor of FGF-receptor tyrosine kinases 1, 2 and 3, inhibits the growth of FGF-receptor 2 driven gastric cancer models in vitro and in vivo
Authors: Xie Liang , Su Xinying , Zhang David , Tang Lily , Xu Jessie , Wang Maggie , Yin Lucy , Zhang Jingchuan , Ye Katherine , Wang Zengquan , Kilgour Elaine , Ji Qunsheng
Published: June 2012

Gastric cancer is the second most lethal cancer worldwide. We and others have shown that the FGFR2 gene is amplified in a subset of gastric cancers. To validate FGFR2 as a potential therapeutic target for gastric cancer, we employed an inducible shRNA system to specifically block expression of individual FGFRs, both in vitro and in vivo. When FGFR2 was specifically down regulated in an FGFR2-amplified gastric cancer cell line Snu-16, the activation of pFGFR2 and its downstream signaling effectors were blocked and there was significant inhibition of the cell growth and survival that further translated into tumor growth regression in vivo, demonstrating that in this model tumor growth/survival is dependent on the FGFR2 pathway. To further confirm this, AZD4547, a selective inhibitor of FGFR 1, 2 and 3 receptor tyrosine kinases was tested in a panel of gastric cancer cell lines for its anti-proliferative effect. In this panel the cell-lines that were most sensitive to AZD4547 were the Snu-16 and KATOIII, both of which are gene amplified for FGFR2. Exposure of the Snu-16 cells to AZD4547 resulted in cell-death and once daily oral dosing of AZD4547 resulted in tumor growth inhibition of the Snu-16 xenografts in a dose-dependent fashion, with significant tumor regression observed when AZD4547 was dosed once daily at 12.5 mg/kg. The anti-tumor efficacy of AZD4547 correlated with inhibition of pFGFR2 and modulation of other downstream markers of FGFR pathway activation, including pErk, pS6 protein and pPLCγ and efficacious doses of AZD4547 induced elevations of levels of plasma FGF23, a known marker of FGFR inhibition. In addition to the single-agent efficacy, combining of AZD4547 with cytotoxic agents, currently used for the treatment of gastric cancer patients, revealed enhanced growth inhibition of Snu-16 xenografts. Taken together, these data support clinical testing of AZD4547 in gastric cancer patients harboring FGFR2 gene amplification.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1643. doi:10.1158/1538-7445.AM2011-1643
Abstract 2823: TAK-441, a novel investigational small molecule hedgehog pathway inhibitor for use in cancer therapy
Authors: Tojo Hideaki , Shibata Sachio , Satoh Yoshihiko , Kawamura Mihoko , Inazuka Masakazu , Yamakawa Hiroko , Kashiwagi Masahide , Miyamoto Maki , Kondo Shigeru , Oohashi Tomohiro , Oguro Yuya , Sasaki Satoshi
Published: June 2012

Dysregulation of hedgehog (Hh) signal, which is caused by Hh-ligand overexpression or patched1 (Ptch1) mutation, has been found to play an important role in tumorigenesis. TAK-441 is a potent and selective smoothened (Smo) antagonist that blocks Hh signaling. In the Gli-responsive promoter-luciferase (Gli-luc) reporter assay in NIH3T3/Gli-luc cells, TAK-441 inhibited Gli transcriptional activity with an IC50 of 4.4 nM. TAK-441 also inhibited expression of human Gli1 messenger mRNA with an IC50 of 1.9 nM in MRC5 human embryonic fibroblasts. In addition, TAK-441 inhibited binding of cyclopamine, a plant-derived well-known Smo inhibitor, to human Smo on 293T cells overexpressing human Smo with an IC50 of 8.6 nM. These data suggest that TAK-441 inhibits the Hh signaling pathway through its binding to Smo.

Oral administration of TAK-441 (0.5, 1, 5, 25 mg/kg QD) in mice bearing a Ptch1 (+/-), p53(−/-) medulloblastoma allograft resulted in dose-dependent antitumor activity with concomitant reduction of Gli1 mRNA expression in the tumors. Complete remissions were observed in the 25 mg/kg QD dose cohort after two consecutive weeks of treatment. Significant (p≤0.025 by one-tailed Williams test) antitumor activity was also detected in sonic hedgehog expressing human primary pancreatic and ovarian cancer xenografts when dosed at 10 and 25 mg/kg QD for two weeks, and in a colon cancer xenograft when dosed at 6.25 and 25 mg/kg BID for three weeks. In these models, the expression of tumor-associated mouse stromal Gli1 mRNA was markedly decreased following TAK-441 treatment, suggesting that Hh signaling in these tumors was driven in a paracrine mode of action. The combination of TAK-441 and rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), demonstrated significantly better antitumor activity than either agent alone in human primary pancreatic cancer xenograft models (p≤0.05 by Students’ t-test). In addition, delayed tumor re-growth was observed in the TAK-441/rapamycin combination treatment group compared with the rapamycin single-agent treatment group after terminating treatment. As a result of these preclinical studies, TAK-441 has recently entered Phase I clinical studies in cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2823. doi:10.1158/1538-7445.AM2011-2823
Abstract 3270: Combination strategy to circumvent the acquired resistance to VEGF receptor tyrosine kinase inhibitor in M24met melanoma in mice
Authors: Zhang Lianglin , Lira Maruja E. , Ching Keith A. , Chen Enhong , Lu Tina , Christensen James G. , Hu-Lowe Dana D.
Published: June 2012

Anti-angiogenic therapy targeting the tumor vasculature has demonstrated clinical benefits in certain types of tumors. However, many patients develop resistance and eventually progress on anti-angiogenic therapy. The mechanism of resistance is largely unknown and warrants extensively investigation. We developed a human xenograft tumor model (M24met/R) in mice that has acquired resistance to VEGF receptor tyrosine kinase inhibitors (VEGFR TKI), by chronically treating the parental M24met tumors (M24met/P) with PF-0337210 (20-40 mg/kg, QD, PO) and serially passaging resistant tumor fragments in nude mice. M24met/R tumors were also resistant to axitinib, a selective VEGFR TKI in late stage clinical development. Immunohistochemical (IHC) staining revealed higher degree of hypoxia and lower microvessel density in M24met/R tumors compared to parental tumors. Compared with those in M24met/P, vessels in M24met/R tumors appeared to be larger in diameter and more mature as shown by high incidence of co-localization between endothelial cells and perivascular cells (CD31+Desmin+). Microarray analysis of tumor lysates (human UA133 2.0, mouse 430 2.0 chip) revealed distinct gene expression profiles between M24met/R tumors and size-matched M24met/P tumors. Of note, several angiogenesis-related genes, including human VEGF, bFGF, integrin α5β1, IL-8, PDGF,CSF and murine VEGF, integrin α5β1, ALK1 (activin receptor-like kinase 1), IL-8, c-Met, IL-6 were up-regulated in M24met/R tumors, whereas murine VEGFR1 and 2, PROX1, PDGFR and endogenous angiogenic inhibitor TSP-1 were down-regulated in the resistant tumors. The above observations were verified with ELISA and/or IHC. Next, targeted combination approaches were applied in an attempt to circumvent M24met/R resistance to axitinib. Concurrent administration of axitinib plus either anti-IL-8 monoclonal antibody (mAb), c-Met inhibitor, anti-integrin α5 mAb or anti-ALK1 mAb significantly improved anti-tumor efficacy compared to single agents alone. IHC analyses showed that combinatorial therapy of anti-ALK1 plus axitinib reduced the amount of CD31+Desmin+ vessels compared to axitinib alone, suggesting that targeting ALK1 destroyed VEGFR TKI-resistant vessels. Together, our results suggested that multiple alternative angiogenic factors contributed to the acquired resistance of M24met/R melanoma to VEGFR TKI. Combination treatment using inhibitor of IL-8, c-Met, integrin α5β1 or ALK1 with VEGFR TKI may represent a new strategy to mitigate resistance to VEGFR TKI in the clinic.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3270. doi:10.1158/1538-7445.AM2011-3270
Abstract 332: B-Raf mutations are associated with a worse outcome in ovarian cancer
Authors: Libra Massimo , Russo Alessia E. , Pellizzari Ilenia , Bagnoli Marina , Raspagliesi Francesco , Sorio Roberto , Baldassarre Gustavo , Canevari Silvana ,   M.I.T.O. Group
Published: June 2012

Epithelial ovarian cancer is the leading cause of death from gynecologic malignancies and its frequency has increased dramatically in the last decade. Most women present with advanced disease at diagnosis due to the insidious onset of this disease and the lack of effective early detection methods. Current treatments, consisting of debulking surgery and subsequent platinum-based chemotherapy, yield a response rate of over 80%, but almost all patients relapse or develop resistance to therapy and ultimately succumb to their cancer. Therefore, there is a need for understanding the molecular pathways involved in ovarian cancer progression and chemoresistance in order to control/reverse them and develop more tailored therapies.

Disregulation of Ras/Raf/MEK/ERK pathway plays a key role in pathogenesis of several human cancers. Mutations at membrane receptors, upstream signalling transducers, as Ras and B-Raf, as well as in genes regulating Raf activity (e.g., PI3K, PTEN, Akt), promote constitutive ERK signaling, stimulating proliferation and survival and providing essential tumour growth and maintenance functions. Conflicting data have been generated about B-Raf mutational status in ovarian cancer. Furthermore, no previous investigations have explored the association of this gene alteration with clinical response.

To this aim, in the present study, tumor samples from 48 advanced ovarian cancer patients, with clinical follow-up of at least 5 years, were analyzed for B-Raf mutations within exon 15. All patients were treated with standard therapy containing paclitaxel and carboplatin chemotherapeutic agents and clinical response was obtained in 58% of them. B-Raf mutation (V600E) was detected in 18 out of 48 (37.5%) patients. Interestingly, this mutation occurs mostly among non responders patients (75% vs 11%; p<0.0001).

These preliminary data indicate that V600E B-Raf mutation may correlate with a loss of clinical response to therapy. To explore this possibility, functional experiments have been also performed by analyzing several ovarian cell lines transfected with mutated B-Raf (V600E). In agreement with our clinical observations, transfected clones showed a higher chemo-resistance, supporting the notion that alteration of Ras/Raf/MEK/ERK pathway may be associated with resistance to chemotherapy. Overall, our data indicate that in ovarian cancer the Ras/Raf/MEK/ERK pathway may represent a promising therapeutic target to reverse chemoresistance.

This work was partially supported by AIRC grants and Italian League against Cancer

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 332. doi:10.1158/1538-7445.AM2011-332
Abstract 3535: Diindolylmethane potentiates the effect of cisplatin in ovarian cancer cells by inhibiting STAT-3 and VEGF
Authors: Kandala Prabodh K. , Wright Stephen E. , Srivastava Sanjay K.
Published: June 2012

Ovarian cancer remains the second leading gynecological cancer in United States. It is usually detected in late stages with poor prognosis. Cisplatin is a chemotherapeutic drug currently used to treat patients with ovarian cancer. However, ovarian tumors develop resistance and cisplatin is associated with systemic toxicity. We previously reported that Diindolylmethane (DIM), a natural compound present in cruciferous vegetables inhibits the proliferation of ovarian cancer cells [Kandala and Srivastava, Mol. Pharmacol. 2010, 78, 297-309]. In this study, we evaluated the effect of DIM with cisplatin in ovarian cancer cells. Exposure of SKOV-3 cells to 50μM DIM for 24 hours followed by treatment with 10µM cisplatin resulted in about 70% reduction in the survival of cells as compared to 30% by cisplatin treatment alone. Western blot analyses indicate that combination (DIM + cisplatin) treatment block the activation of STAT-3 by inhibiting the phosphorylation at Tyr 705 and Ser 727 as compared to cisplatin or DIM treatment alone. The STAT-3 protein level was reduced by 90% by combination treatment. Mcl-1and survivin, which are regulated by STAT-3 were also down-regulated by combination treatment. Interestingly, survivin was up-regulated by cisplatin treatment but completely blocked by combination treatment. Increased apoptosis by combination treatment was evident by caspase-3 and PARP cleavage. In addition, we tested the effects of this combination treatment on ovarian tumor angiogenesis and metastatsis. The results of aortic ring assay suggested that the combination treatment completely suppressed the formation of microvessel sprouts. Furthermore, combination treatment blocked invasion of cells by approximately 50% as evaluated by Boyden's chamber. Combination of DIM and cisplatin suppressed VEGF secretion by 50% as compared to 20% by cisplatin treatment in SKOV-3 cells. Taken together, our results suggest that DIM potentiates the effect of cisplatin by blocking the activation of STAT-3 and inhibiting angiogenesis and metastasis. Further mechanistic and in vivo testing of this combination treatment is under progress. [Supported in part by R01 grants CA106953 and CA 129038 (to S.K.S) awarded by the National Cancer Institute].

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3535. doi:10.1158/1538-7445.AM2011-3535
Abstract 3592: Preclinical combination strategies for development of JI-101, a selective inhibitor of Ephrin B4, VEGF R2 and PDGF-Rβ
Authors: Gilcrease Glynn W. , Wade Mark , Mullangi Ramesh , Srinivasa Sreesha , Northrup Jonathan , Govindarajan Raman , Sharma Sunil
Published: June 2012

Background: JI-101 is a novel, highly selective and potent angiogenesis inhibitor with unique EphB4 activity along with VEGFR2 and PDGFRβ inhibitor activity. JI-101 shows a good pre-clinical profile and is well tolerated to date in phase 1 and early phase 2 clinical trials. In order to investigate combination partners, in vitro and in vivo studies were undertaken to combine JI-101 with chemotherapies and various targeted agents.

Materials and Methods: Cancer cell lines from different tissue types were seeded on 96 well plates at a density of 2,000 cell per well and allowed to adhere overnight. To determine if JI101 could enhance the toxic effects of traditional chemotherapy, cells were incubated with 10-0.003 μM Gemcitabine, Oxaliplatin and 5-Fluorouricil in the presence or absence of 1 μM JI101. To determine the effect of JI101 with other targeted therapies, cells were incubated with 10-0.03 μM JI101, Axitininb, and Imantinib and 1-0.003 μM BEZ235. In addition, cells incubated with BEZ235 were combined with 1 μM of JI101, Axitininb or Imatinib. Cells were incubated with the drugs for 96 hours at 37oC in a cell culture incubator. After incubation cell viability was tested with the MTS Assay.

Results: Toxicity profiles of traditional chemotherapies were not significantly altered when combined with JI101. Incubation of BEZ235 with multiple cancer cell lines resulted in dose dependent toxicity to the cells whereas JI101, Axitinib and Imatinib showed little to no toxicity in the cancer cell lines tested. Coincubation of the PI3 kinase/mTOR inhibitor BEZ235 with JI101, an EphB4, VEGFR2, and PDGF-Rβ or Axitinib a specific VEGF-R2 inhibitor enhanced the cellular toxicity of BEZ235 in a cell line specific manner.

Conclusions: JI-101 is highly synergistic with mTOR inhibitors and PI 3 kinase inhibitors in vitro and in vivo. The molecular mechanism will be presented for this synergy. A combination trial of JI101 and PI3 kinase inhibitor or JI101 and mTOR inhibitor is indicated.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3592. doi:10.1158/1538-7445.AM2011-3592
Abstract 3680: Comparison of risk of advanced cervical lesions among women with first detection of atypical squamous cells, atypical glandular cells, and low-grade squamous intraepithelial lesions by Pap smear
Authors: Chen Yun-Yuan , You San-Lin , Koong Shin-Lan , Chen Chi-An , Chen Chien-Jen ,   Taiwan Cervical Cancer Screening Task Force
Published: June 2012

The study aimed to examine the incidence of invasive cervical cancer (ICC), cervical carcinoma in situ (CIS), and cytologic high-grade squamous intraepithelial lesions (HSIL) or more severe cytologic lesions among women detected their first atypical squamous cell undetermined of significance (ASC), atypical glandular cells (AGC), and low-grade squamous epithelial lesions (LSIL), and to estimate the associations between the subsequent follow-up attending and risks of developing cervical cancer.

National cervical cancer screening program in Taiwan has been implemented since 1995, all screening records are mandatory to report, and a centralized screening database has been established as well. The first cytologic abnormalities, included 110,561 ASC cases, 8,339 AGC cases, and 45,671 LSIL cases, and cytologic HSIL or above lesions were identified from Taiwan cervical cancer screening registry. Women received Pap smear or biopsy in one year after their first cytologic abnormalities were considered have attended subsequent follow-up. Cervical cancers were ascertained from national cancer registry in Taiwan until Dec, 31st, 2007. Hazard ratios were estimated by Cox's proportional hazards model, and cumulative incidences were estimated by Nelson-Aalen method.

There was at least 14% reduction of CIS and 23% reduction of ICC if women attended screening two years before their first abnormalities, and at least 32% reduction of CIS and 58% reduction of ICC if they attended screening within two years. Follow-up attending for the first abnormality significantly reduced 13% to 53% of cytologic HSIL occurrence, 40% to 49% of CIS occurrence, and 52% to 71% of ICC occurrence. AGC is the most clinical significant lesion compared with ASC and LSIL, and is suggestive to include in the positivity follow-up system. Compared to LSIL, ASC is less risky to develop cytologic HSIL, similar risk to develop CIS, and possesses more risk to develop ICC. Subsequent follow-up attendance for their first abnormality and previous negative smear detection were inversely associated with cervical cancer occurrence.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3680. doi:10.1158/1538-7445.AM2011-3680
Abstract 3790: Loss of BRCA1 regulates autophagy as a cellular stress response in breast and ovarian cancer cells
Authors: Tang Maggie Kei Shuen , Wong Alice Sze Tsai
Published: June 2012

Inactivation of cell death is a major step in tumor development. The BRCA1 tumor suppressor, which is frequently mutated in human breast and ovarian cancers, is a critical mediator of cell death. Although the role of BRCA1 gene loss for inhibiting apoptosis is well established, little is known regarding its involvement in other cell survival pathways. Increasing evidence strongly supports that autophagy is a key strategy for cell survival in response to stress. Endoplasmic reticulum (ER) stress and nutrient deprivation are common environmental stresses governing the survival and adaptation of tumor cells in vivo. Here we show that BRCA1 is an important regulator of autophagy, particularly under stress conditions. We found that autophagy could be induced by chemicals that cause ER stress and nutrient deprivation in T47D breast cancer cells and OVCAR-3 ovarian cancer cells with targeted inhibition of endogenous BRCA1 using small interfering RNA. This was characterized by conversion of microtubule-associated protein 1 light chain 3 (LC3) I into LC3 II, punctate green fluorescent protein-labeled LC3 fluorescence, and autophagosomes formation. Treatment of cells with the lysosomal inhibitors, Pepstatin A and E-64d, augmented autophagy, suggesting that autophagosomes were actively produced. In contrast, BRCA1-mediated autophagy that was induced under stress conditions was inhibited by treatment with autophagy inhibitor 3-methyladenine. Similar results were obtained in cells with decreased beclin 1 level by RNA interference. These results reveal a novel link between BRCA1 and autophagy, which may contribute to the survival of BRCA1-deficient cancer cells in their microenvironment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3790. doi:10.1158/1538-7445.AM2011-3790
Abstract 380: Magnetic nanoplatforms for tumor targeting, imaging and energy delivery
Authors: Sridhar Srinivas , Campbell Robert , Nagesha Dattatri , Gultepe Evin
Published: June 2012

We have developed magnetic nano-liposomes (MNL), incorporating superparamagnetic iron oxide nanoparticles (SPION), that are versatile theranostic nanoplatform for enhanced drug delivery and monitoring of cancer treatment. MNL are prepared with a formuation of DPPC:DOTAP:CHOL and DOPE-PEG5000. Incorporation of SPIONs results in MNL with mean diameter of 150-250 nm. MNL are easily taken up by B16-F10 melanoma, HUMVEC-D and breast cancer cell lines. They preferentially target the tumor vasculature as shown in a dorsal skin fold chamber using fluorescently labeled MNL. MNL display superparamagnetic response that is essential for magnetic targeting, MR contrast enhancement and magnetic heating.

MNL was administrated to SCID mouse with metastatic (B16-F10) melanoma grown in the right flank. Pre-injection and post-injection MR images were used to assess response to magnetic targeting effects. Biodistribution studies were conducted by 111In labeled MNL and amount of radioactivity recovered was used to confirm the effect of targeting for intratumoral administrations.

We have shown that tumor signal intensities in T2 weighted images decreased an average of 20±5% and T2* values decreased and average of 14±7ms in the absence of magnetic targeting. This compares to an average signal decrease of 57±12% and a decrease in T2* relaxation times of 27±8ms with the aid of external magnet showing up to 2-fold greater accumulation by magnetic targeting.

111In radio-labeled MNL have been shown to enable multi-modal imaging in vivo using MRI and SPeCT/CT. The images show that an MNL bolus injected intra-tumorally was retained in the tumor 24 hours after injection. Application of a magnetic field enables redistribution of the MNL in the tumor.

These MNL are also responsive to ac magnetic fields applied using a Copper coil at 360 kHz and 170A driving current. Both hyperthermia (upto 45C) and thermo-ablative temperatures upto 90C were achieved in 10 – 30 minutes ex vivo in buffer. The results indicate high efficiency for magnetic heating using MNL (Specific Absorption Rate ∼ 104 W/kg) and demonstrate the capability to couple ac magnetic fields to MNL to achieve any set of temperatures needed for hyperthermia and thermal ablation.

To date there are no truly theranostic platforms that have been approved for clinical use that combine targeting, thermal heating and MRI imaging capabilities. Existing FDA approved magnetic nanoparticle formulations like ferridex and ferumoxytol, are optimized for MR imaging, and have not been shown to be usable for thermal therapy.The MNL platform is a novel nanoplatform combining multi-modal imaging capabilities, with magnetic targeting and thermal therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 380. doi:10.1158/1538-7445.AM2011-380
Abstract 4431: Antitumor activity of nanosponge-encapsulated camptotechin in human prostate tumors
Authors: Minelli Rosalba , Cavalli Roberta , Fantozzi Roberto , Dianzani Chiara , Pettazzoni Piergiorgio , Ellis Leigh , Shen Li , Pili Roberto
Published: June 2012

Drug delivery technologies are new and powerful approaches to enhance the pharmacological properties of drugs by improving their availability and stability and by reducing their side effects.

Camptothecin (CPT) is a potent DNA Topoisomerase I inhibitor that presents strong antitumoral efficacy in both hematological and epithelial malignancies. However, its use is limited because of its high degradability and weak solubility. Indeed, the structure of camptothecin presents a lactone ring that is subjected to hydrolysis in physiological conditions with consequent inactivation of the topoisomerase binding activity. In the present study a new formulation of CPT embedded in cyclodextrin-based nanosponges (CN-CPT) has been evaluated in vitro and in vivo on prostate cancer models. CN-CPT manifested a strong antitumoral efficacy revealed by MTT and colony forming assay on hormonal independent PC3 and DU145 cells, significantly higher as compared to the same dose of free CPT. Moreover, cell cycle analysis and annexin V/PI staining showed that CN-CPT treatments induced significant apoptotic cell death at 1 nM (61.7% apoptotic cell death; p<<0.001), where free CTP (4.36% apoptotic cell death) was not effective. The efficacy of CN-CPT has been tested in vivo in PC3 and DU145 xenograft experiments that have confirmed a significant improvement of antitumoral activity of CN-CPT as compared to CPT. To evaluate possible antimetastatic activity of CPT, in vitro experiments of tumor cell adhesion to HUVECs as a model of endothelium and cell motility were performed. Results showed that CN-CPT is effective at lower doses compared to CPT in inducing inhibition of adhesion and motility. In order to evaluate whether androgen receptor signaling could affect the responsiveness to either CPT or CN-CPT the AR expressing murine models of prostate cancer (MYC-AD, PTEN8 and PTEN CAP8) have been used. Results showed that expression of functional AR diminished the efficacy of both C

PT and CN-CPT. However the latter still exerted greater effects compared to CN-CPT. Taken together these results support the use of CPT and the development of nanotechnologies for drug delivery in the treatment of castrate refractory prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4431. doi:10.1158/1538-7445.AM2011-4431
Abstract 4461: MPT0B098, a novel microtubule inhibitor, displays potent anti-angiogenic activity via destabilizing hypoxia-inducible factor-1alpha mRNA
Authors: Cheng Yun-ching , Liou Jing-Ping , Lai Wen-Yang , Chang Chi-Yen , Pan Wen-Yu , Kuo Ching-Chuan , Chang Jang-Yang
Published: June 2012

Identification of a single agent that is able to target tumor cells, rapidly destroy tumor vasculature and inhibit angiogenesis could be an ideal approach for targeting solid tumors. We recently discover a novel sulfonamide compound, 7-aryl-indoline-1-benzene-sulfonamide (MPT0B098), as a potent microtubule inhibitor through binding to the colchicine binding site of tubulin. MPT0B098 is active against various human cancer cell growth with IC50 values ranged from 70-300 nM. Notably, HUVEC cells exhibit less susceptibility to the inhibitory effect of MPT0B098 with an IC50 of 510 nM. In addition, MPT0B098 exhibits no cross resistance with vincristine, paclitaxel, etoposide and camptothecin-resistant cell lines. Interestingly, MPT0B098 is still active toward cells (KB L30) with beta1-tubulin mutation. MPT0B098 arrests cells in G2/M phase and subsequently induces cell apoptosis through the caspase-dependent apoptotic pathway. In addition, using sub-lethal dose, MPT0B098 effectively suppressed to the tube formation and migration of HUVECs induced by VEGF. The expression levels of HIF-1alpha and VEGF were significantly inhibited in a concentration-dependent manner by MPT0B098 under hypoxic condition. Furthermore, HIF-1alpha-regulated genes, including cathepsin D, PDGF, VEGF, and VHL, were found to be down-regulated by MPT0B098 in A549 cells. Moreover, the decrease of the amount of HuR, a HIF-1alpha mRNA stability protein, translocated from nuclear to cytoplasm and suppression of AKT activity were coincided with decreased amount of HIF-1alpha mRNA by MPT0B098 treatment under hypoxia condition. MPT0B098 significantly suppressed tumor growth and microvessel density of tumor in H460 and KB-Vin10 xenografted mouse model. Taken together, our results indicate that MPT0B098 is a promising anticancer drug with potential for the treatment of human malignancies. (The study was supported by grants of DOH99-TD-C-111-004 from Department of Health and CA-099-PP-02 from National Health Research Institutes, Taiwan, R.O.C.)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4461. doi:10.1158/1538-7445.AM2011-4461
Abstract 4585: Targeting of CD5 on chronic lymphocytic leukemia cells with antibody mixtures
Authors: Klitgaard Josephine L. , Geisler Christian , Pedersen Mikkel W.
Published: June 2012

Chronic Lymphocytic Leukemia (CLL) is the most common form of leukemia found in adults in Western countries. Approximately 2 out of every 100,000 people develop CLL annually, with 90% being older than age 50. CLL arises from accumulation of CD5+ B lymphocytes. CLL cells have defective apoptotic pathways and are characterized and distinguished from other leukemic cells by expression of CD19, CD20, and CD23 along with CD5. Current targeted therapy for treatment of CLL includes monoclonal antibodies against CD20 or CD52. However, these are often either not very effective against CLL cells due to low target expression, or are associated with severe side effects due to expression of target on other immune cells. Thus, more specific and efficacious treatments of CLL are warranted.

Due to its relatively specific expression on CLL cells, CD5 is an obvious target for antibody based therapeutics. However, earlier attempts to develop antibodies against CD5 for treatment of CLL have failed to show significant therapeutic activity in patients. As in vivo studies with murine antibodies against CD5 showed no direct effect of the antibodies, activation of secondary effector functions might be important to obtain therapeutic activity with antibodies targeting CD5. Recently, it has been shown that mixtures of monoclonal antibodies against the same target increase complement-dependent cytotoxicity. Hence, it could be relevant to target CD5 with more than one monoclonal antibody to increase activation of complement-dependent cytotoxicity.

In the current work, sixty-eight chimeric IgG1 antibodies against human CD5 were cloned from CD5 immunized mice using our mSymplex™ technology. Fifteen unique clonotypes were selected for further characterization and functional analyses. The selected antibodies were epitope binned using Biacore analysis to identify antibodies with non-overlapping epitopes. Using confocal microscopy, the level of internalization of CD5 upon binding of the single antibodies and antibody mixtures to CD5 was investigated and it was shown that some combinations of two non-overlapping antibodies induce internalization of CD5, while mixtures of two other non-overlapping antibodies remain on the surface bound to CD5. Subsequently, the direct cytotoxic effect of CD5-specific antibody mixtures and the ability to mediate ADCC and CDC was investigated in vitro using CLL cells isolated from human patients. An update will be presented.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4585. doi:10.1158/1538-7445.AM2011-4585
Abstract 4780: c-FLIP knockdown increases apoptosis in cervical cancer cell lines
Authors: Ili Carmen , Brebi Priscilla , Garcia Patricia , LeBron Cynthia , Leal Pamela , Montenegro Sonia , Corvalan Alejandro , Guerrero-Preston Rafael , Roa Juan Carlos
Published: June 2012

Cervical Uterine cancer is a leading cause of cancer deaths in women worldwide. Its natural history begins gradually from low-grade and high-grades squamous intraepithelial lesions to an invasive disease. Decades of studies have confirmed a directly association between persistent high-risk Human Papillomavirus (HPV) infections and cervical cancer. However, high-risk HPV infections are not sufficient to induce cellular transformation and tumor occurrence. The cellular FLICE-like inhibitory protein (c-FLIP) has been founded overexpressed in several cancers and could be associated with cervical carcinogenesis. c-FLIP is a catalytically inactive caspase-8/-10 homologue, and when is overexpressed has the ability to inhibit the extrinsic pathway of apoptosis. Aim: To characterize c-FLIP participation in cervical carcinogenesis.Materials and Methods: Four cell lines were used in this study: ECT1 E6/E7 (squamous normal cervical epithelial), SiHa (cervical carcinoma containing an integrated HPV 16 genome), C-4I (cervical carcinoma containing an integrated HPV 18 genome) and C-33A (cervical carcinoma without HPV). c-FLIP expression in cell lines was determined by real time PCR and western blot. c-FLIP expression was transiently downregulated by siRNA directed against c-FLIP mRNA and the silencing effects on cell viability and apoptosis were analysed, by comparing with a control negative siRNA-transfected cells.Results: c-FLIP was found to be overexpressed in SiHa and C-4I cell lines and repressed in C-33A cell line. Only the long variant of c-FLIP was found in cervical cancer cell lines. Transient knockdown of c-FLIP resulted in significant reduction in cell viability (p< 0.001), compared with negative control siRNA-transfected cells. Knockdown cells lines exhibited an increased apoptotic rate in comparison with controls (p<0,001). Conclusions: The long variant of c-FLIP is overexpressed in cervical cancer cell lines with integrated genome of a high-risk HPV. Furthermore, the siRNA-mediated downregulation of c-FLIP expression induces apoptosis and decreases cell viability in cervical cancer cell lines. Therefore, this protein could be a potential therapeutic target in cervical cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4780. doi:10.1158/1538-7445.AM2011-4780
Abstract 5088: Exploring ATM deficiency as a potential therapeutic target for the PARP inhibitor olaparib in head and neck squamous cell carcinoma
Authors: Senra Joana M. , Donald Emma L. , Williams Victoria S. , Wombwell Helen , Hall Gillian , Harbron Chris G. , Telfer Brian A. , Morten John , Womack Chris , West Catharine M. , Hodgson Darren , O'Connor Mark J. , Wedge Steve R. , Stratford Ian J.
Published: June 2012

Background: Ataxia telangiectasia mutated (ATM) gene, localized in chromosome 11q22-23 is a key player in the signaling response to double strand breaks (DSBs). Impaired DSB repair due to ATM deficiency in tumor cells may lead to a wider application of the ‘‘synthetic lethality’’ approach seen with PARP inhibitors in BRCA-deficient tumors. Targeting DNA repair mechanisms with specific small molecule inhibitors will in principle sensitize tumor cells to DNA-damaging agents such as radiation. Radiation therapy remains the most common treatment of choice for head and neck cancers; however, there is still a large scope for improvement. Loss of distal 11q chromosome and subsequent ATM deficiency has been reported in primary head and neck tumors. Therefore, there are two aims to this work. First, we will establish the potential of targeting this cellular deficiency with the potent poly (ADP-ribose) polymerase (PARP) inhibitor olaparib; second, we will determine the prognostic significance of ATM deficiency in patients with head and neck squamous cell carcinoma (HNSCC) undergoing radiotherapy.

Methods: A panel of 10 HNSCC cell lines was screened for sensitivity to radiation in combination with olaparib by clonogenic survival assay in normoxic and anoxic conditions. The effect of olaparib as a single agent was also assessed in a PE/CA PJ34 (ATM deficient) xenograft model. A panel of 155 HNSCC biopsies taken prior to treatment with radiotherapy and where 5 year outcome data was available were used for ATM expression analysis by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH).

Results: Olaparib enhanced the effect of ionizing radiation (IR) in all HNSCC cell lines. In vitro, the increase in sensitivity to 2Gy after olaparib treatment ranged between 1.5- and 8.4-fold in normoxia and between 1.2- and 3.9-fold in anoxia. In vivo, administration of olaparib (50mg/kg p.o) as a single agent in PE/CA PJ34 xenografts daily for 5 days caused a substantial increase in tumor growth delay compared to vehicle alone. The IHC and FISH studies of the 155 HNSCC biopsies showed a variable level of ATM deficiency and the prognostic significance (if any) of these observations are being established.

Conclusion: We demonstrate that olaparib is a promising candidate as a single agent in tumors with ATM deficiency, and given the radiosensitizing effects in vitro and in vivo it warrants consideration for combining with radiation for the treatment of HNSCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5088. doi:10.1158/1538-7445.AM2011-5088