bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 704: Development of therapeutic antibodies targeting Notch3 in lung cancer
Authors: Lin Luping , Mernaugh Raymond , Carnahan Robert , Carbone David P. , Dang Thao P.
Published: February 2011

The Notch receptors are essential for both normal development and tumorigenesis in many human cancers. Notch3 is expressed in 40% of all lung cancers. Inhibiting this pathway results in reduced tumor growth in lung in vitro as well as in vivo. Thus, this pathway represents a potentially important target for therapeutic development. Here we report the early results of a strategy to inhibiting Notch3 signaling through the development of Notch3 monoclonal antibodies. Using a Notch3 peptide library, we discovered two regions of the extracellular domain, believed to be the binding regions for the Notch3 ligand, Jagged1. The recombinant proteins representing these two regions, respectively, were used as antigens to immunize mice. We demonstrated that antisera from these mice immunized with portions of the receptor extracellular domain can inhibit Notch3 activation. Further developments of hybridoma clones were screened with ELISA, immunoprecipitation and their ability to inhibit Notch3 cleavage. 12 hybridoma clones were selected, and 4 out of 12 antibodies with IgG1 or IgM isotypes were found to specifically inhibit Notch3 activation but not Notch1. Further testing is on going to validate these findings as well as to determine affinity and anti-tumor activity of these antibodies. Development of monoclonal antibody to specifically inhibit Notch3 ligand binding regions could serve as a strategy for the development of future therapeutics for patients with lung 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 704.
Abstract 71: Metformin inhibits lung cancer cell growth and angiogenesis in vitro and in vivo: Effect on IL6-Stat3 pathway
Authors: Lin Chien-Chung , Shen Mei-Chun , Su Wu-Chou
Published: February 2011

The usage of metformin as a first line treatment for type 2 diabetes associates with reduced cancer risk. Animal studies have shown that metformin inhibits breast cancer growth mainly via AMPK-mTOR pathway. Associated clinical trial is conducting. However, the application of metformin in other cancers and other mechanisms remain not clarified. In our previous study, we demonstrated the down-regulation of TSC1 (tuberous sclerosis 1) in lung cancer cells from malignant pleural effusion (MPE) and verified with qRT-PCR and immunohistochemistry. TSC1/TSC2 complex can suppress the mTOR pathway and receive input from LKB1-AMPK pathway and we found that metformin induced TSC1expression in vitro. Previous studies demonstrate that TNFα-sphingomyelinase and IL6-Stat3 contribute the formation of MPE via activating VEGF secretion. TNF-α can also induce IL6 secretion in many tumors. In our recent study, we first investigated the effect of metformin on proliferation of lung cancer cell. Lung cancer lines including PC14PE6/AS2 (AS2), A549 and CL 1-0 were treated with various dosage of metformin. After treatment for 96 hours, metformin inhibited proliferation of AS2, A549 and CL1-0 lung cancer cells dose-dependently. The IC50 of all cells are around 2.5-5 mM, measured by MTT assays. We also investigated the addictive cytotoxicity of metformin to cisplatin. Metformin co-incubation with cisplatin for 72 hr in AS2 cells displayed an increased cytotoxicity as compared to each agent alone. As for the association with angiogenesis, by analyzing the supernatant with ELISA assay, the VEGF secretion also decreased after treatment with metformin in AS2 and A549 cells. And Western blotting was conducted to access the effect on Stat3 and AMPK phosphorylation of metformin. In AS2 cell, not only the phosphorylation of AMPK increased after metformin treatment, the Stat3 phosphorylation was also suppressed. In addition, stimulation with TNF-α led to substantial secretion of IL6, which was inhibited by co-incubation with metformin. In vivo, the AS2 cells were injected i.p. into the unanesthetized nude mice and MQ-water or metformin (1mg) were injected 2 days later. Around 2 to 4 weeks, the mice developed peritoneal carcinomatosis and malignant ascites. In the metformin group, though the same number of mice developed peritoneal carcinomatosis and ascites, the amount of ascites and VEGF level are less than the control group. In conclusion, metformin inhibit lung cancer cell proliferation and angiogenesis in vitro and in vivo. And the IL6-Stat3-VEGF pathway may also involve in the anticancer effect of metformin.

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 71.
Abstract 712: Antitumor activity of SPINK1 monoclonal antibody in a subset of SPINK1 positive ETS-negative prostate cancer
Authors: Ateeq Bushra , Tomlins Scott A. , Laxman Bharathi , Cao Qi , Cao Xuhong , Jing Xiaojun , Pienta Kenneth J. , Varambally Sooryanarayana , Chinnaiyan Arul M.
Published: February 2011

We have identified SPINK1 outlier expression in an aggressive subset of TMPRSS2:ETS-negative prostate cancers (10% of all prostate cancers) by outlier meta-analysis (meta-COPA). We have further validated the mutual exclusivity of SPINK1 expression and ETS fusion status, demonstrated that SPINK1 outlier expression can be detected non-invasively in urine, and is a useful prognostic marker in prostate cancer (Tomlins et al., 2008 and Laxman et al., 2008). Here we examined the in vitro and in vivo effects of SPINK1 outlier expression. We demonstrate that exogenous SPINK1 recombinant protein stimulate cell proliferation in benign and cancerous prostate cells. Benign prostate epithelial cells RWPE treated with SPINK1 recombinant protein or conditioned medium (CM) from 22RV1 cells (SPINK1+) showed dramatic increase in cell invasion in Boyden chamber Matrigel invasion assay, which was attenuated in the presence of an anti-SPINK1 monoclonal antibody (mAb). We also showed that invasion of 22RV1 cells is attenuated with SPINK1 mAb, while SPINK1 mAb has no effect on the invasion of PC3 cells (SPINK1−) invasion. SiRNA knockdown of SPINK1 in 22RV1 cells attenuated invasion, which could be rescued by recombinant SPINK1 protein or 22RV1 CM. Stable knockdown of SPINK1 in 22RV1 cells using shRNA resulted in decreased cell proliferation, cell invasion and motility. Moreover, stable knockdown of SPINK1 in 22RV1-Luc cells showed reduced tumor burden in an in vivo xenograft model. The preclinical in vivo efficacy of SPINK1 mAb was evaluated in SPINK1 positive 22RV1-Luc xenograft model established in BalbC nu. nu mice. Antitumor activities were determined from the analyses of tumor growth inhibition, defined as the decrease in the mean tumor volume for SPINK1 mAb treated mice versus IgG mAb treated mice. Twice a week intra-peritoneal administration of SPINK1 mAb (10mg/kg) resulted in a 65% reduction of tumor burden. This study provides the rationale for the development of humanized anti-SPINK1 monoclonal antibodies for clinical targeting of the subset of patients with SPINK1 positive and ETS-negative prostate 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 712.
Abstract 72: Metformin enhances tumor regression and reduces tumor burden in a preclinical paradigm modeling obesity's impact on postmenopausal breast cancer
Authors: Giles Erin D. , Anderson Steven M. , Schedin Pepper J. , Thor Ann D. , Johnson Ginger C. , Hedman Karla J. , Mahan M Chad , Houser Jordan L. , MacLean Paul S.
Published: February 2011

BACKGROUND: Obesity and type II diabetes have been linked to a higher risk of breast cancer in post-menopausal women. Patients treated with the anti-diabetic drug metformin (1,1-dimethylbiguanide hydrochloride) for type II diabetes or metabolic syndrome have shown a reduced risk of breast cancer, a greater pathologic complete response for neoadjuvant therapy and an improved breast cancer survival. The purpose of this study was to examine the effects of metformin on tumor promotion in a rodent model of obesity and postmenopausal breast cancer.

METHODS: Rats were injected with N-methylnitrosourea (MNU, 50 mg/kg) at 8 weeks of age to induce mammary tumors, and fed a high fat diet (from 5 weeks of age) to promote obesity. Obesity-resistant and obesity-prone rats were selected, based upon weight gain (between 10 and 14 weeks of age) and were matured under these conditions to produce adult, tumor-bearing lean and obese rats (24±1 vs 29±1 % body fat). Tumors were monitored by manual palpation at weekly intervals from the time of MNU administration. Obese animals were randomly assigned to either metformin treatment (2mg/mL in their drinking water, n=8) or control group (water only, n=6), after at least 1 tumor reached a volume >1cm3. Rats were maintained on their respective treatments for the duration of the study. One week following the initiation of treatment, animals were ovariectomized (OVX) to mimic the post-menopausal state, and tumor palpations were continued for an additional 9 weeks.

RESULTS: Prior to OVX, control and metformin treated obese rats were similar in weight (413±21 vs 397±20 g) and tumor burden (3.3±1.2 vs 3.7±2.3 g). In response to OVX, tumor burden decreased in all animals, as predicted, based on the hormone responsiveness of MNU-induced tumors. Compared to untreated obese rats, the percent of pre-OVX tumor burden was lower in metformin-treated rats (weeks 1-3 post-OVX; 67±10 vs 26± 8%, p<0.05). A tendency for metformin to attenuate the subsequent rebound in tumor burden was observed at week 7 (126±67 vs 41±35%, p=0.06), week 8 (170±112 vs 35±29%, p=0.06), and week 9 (249±165 vs 41±30%, p=0.09).

CONCLUSIONS: These preliminary observations indicate that metformin enhances OVX-induced tumor regression in obese rats and may reduce long term tumor growth and survival in these animals after the surgery. These findings suggest that metformin may have therapeutic benefits in obese, peri- or post-menopausal patients who are at risk for breast 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 72.
Abstract 723: Beyond temsirolimus: Discovery of PKI-587 a highly efficacious dual PI3K/mTOR inhibitor
Authors: Venkatesan Aranapakam M. , Dehnhardt Christoph M. , Santos Efren Delos , Chen Zecheng , Santos Osvaldo Dos , Ayral-Kaloustian Semiramis , Mallon Robert , Hollander Irwin , Feldberg Larry , Lucas Judy , Yu Ker , Brooijmans Natasja , Bard Joel , Gibbons James , Abraham Robert T. , Mansour Tarek S.
Published: February 2011

Temsirolimus, a rapamycin analog, is a selective mTor inhibitor that has been successfully used in the clinic for the treatment of renal cancer. mTOR integrates signals from multiple upstream growth and survival regulatory pathways including the LKB1/AMPK and PI3K (Phosphatidylinositol-3-kinase)/Akt pathways. Among these, Phosphatidylinositol-3-kinase (PI3-K) is a lipid kinase which phophorylates phosphatidylinositol diphosphate (PIP-2) to phosphatidylinositol triphosphate. This pathway regulates cell proliferation, growth, survival, and apoptosis. The aberrant activation of PI3K-α and its downstream effectors including Akt and mTOR, has been linked to the initiation and maintenance of numerous tumor phenotypes. During tumorigenesis, PI3K/Akt/mTOR pathway activation occurs through various mechanisms, including loss of PTEN (the phosphatase that regulates PI3K signaling), over-expression or activation of certain receptor tyrosine kinases (e.g. EGFR, HER-2), interaction with activated Ras, overexpression of the PI3K-α gene (PIKC3A), or mutations in PIKC3A that cause elevated PI3K kinase activity. Deregulated PI3K/Akt/mTOR pathway signaling has been implicated in poor prognosis and low survival rate in patients with various lymphatic tumors, glioblastomas, melanomas as well as breast, prostate, lung, colon, and ovarian cancers. Numerous pre-clinical and clinical studies indicate that PI3K-α plays a key role in the biology of human cancer. Hence, it is likely that a small molecule inhibitor of both PI3K and mTOR will have clinical utility.

Our project team has identified small molecules that potently inhibit the PI3K/mTor pathway. The current clinical candidate, PKI-587 (whose structure will be disclosed) is a pan PI3K (class I)/mTOR inhibitor. PKI-587 decreases tumor cell survival and proliferation in vitro. Additionally, PKI-587 has shown compelling efficacy in multiple human tumor nude mouse xenograft models such as MDA 361 (Breast), U87 (Glioma), H1975, A549 (NSCLC) in nude mice when administered as a single agent. The design of this novel potent inhibitor, synthesis, structure-activity relationships, PK and in vivo efficacy data will be presented.

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 723.
Abstract 724: Selective β2-adrenergic receptor agonists inhibit the proliferation of 1321N1 astrocytoma cells
Authors: Wainer Irving W. , Toll Lawrence , Jimenez Lucita , Polgar Willma , Green Carol , Gow Jason , O'Loughlin Kathleen , Bernier Michel
Published: February 2011

Selective β2-adrenergic receptor agonists inhibit the proliferation of 1321N1 astrocytoma cells

Lawrence Toll1, Lucita Jimenez1, Willma Polgar1, Carol Green1, Jason Gow1, Kathleen O'Loughlin1, Michel Bernier2, Irving W. Wainer2

1 SRI International, Menlo Park, CA

2 Intramural Research Program, National Institute of Aging, Baltimore, MD

We have reported the synthesis of fenoterol analogs and the characterization of these compounds as selective β2-adrenergic receptor (β2-AR) agonists, which have a broad range of abilities to stimulate cAMP accumulation in β2-AR transfected HEK cells, EC50cAMP. We now report the effect of a series of these compounds on the growth of 1321N1 astrocytoma cells (Table). In the 1321N1 cells, β2-AR stimulation by fenoterol analogs potently inhibited [3H]-thymidine incorporation with IC50 values that correlated with the EC50cAMP values. The effects were blocked by the β2-AR antagonist propranolol and mimicked by the adenylate cyclase activator forskolin. (R,R)-Fenoterol induced G1 arrest with an associated decrease in the proportion of cells in G2 and S phase and modulated the levels of proteins involved in cell division. The results indicate that selective β2-AR agonists may exert unanticipated antitumor effects against neural-derived neoplasms that express the β2-AR.Table.Functional activities of fenoterol analogs. Stimulation of cAMP accumulation was determined in intact HEK cells transfected with the β2-AR. Fenoterol analogs do not inhibit mitogenesis in HEK cells. Inhibition of [3H]-thymidine incorporation was conducted in 1321N1 cells in which the β2-AR is natively expressed. Results expressed as mean ± SD, n = 4.CompoundStimulation of cAMP AccumulationMitogenesis Inhibition EC50 (nM)% StimulationIC50 (nM)Isoproterenol0.20 ± 0.091000.05 ± 0.01(R,R)-fenoterol0.30 ± 0.09123.30 ± 8.790.14 ± 0.07(R,R)-4-methoxyfenoterol0.30 ± 0.23135.70 ± 11.030.17 ± 0.02(R,R)-ethylfenoterol2.80 ± 0.90125.40 ± 5.741.44 ± 0.27(R,R)-1-naphthylfenoterol12.5 ± 3.50129.20± 23.61.57 ± 0.34(R,R)-2-naphthylfenoterol0.40 ± 0.1290.30 ± 9.811.91 ± 0.57(R,R)-4-methoxy-1-naphthyl fenoterol3.90 ± 1.80105.90 ± 11.063.98 ± 0.28

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 724.
Abstract 728: Development of pharmacophore-based RET inhibitors for thyroid cancer therapy
Authors: Huang Jui-wen , Ko Ching-Huai , Chen Ting-Shou , Lee On , Tseng Hsiang-Wen , Tsai Yuan-Jang , Liu Chih-Peng , Chen Yen-Chun , Wang Ling-Mei , Hwang Chrong-Shiong
Published: February 2011

The new therapeutic option is pivotal due to the restricted efficacy of current treatments (surgery, radioactive iodine treatment, and chemotherapy) in progressive thyroid carcinomas. Gain-of-function mutations of the receptor tyrosine kinase RET have been identified as driving oncogenic events in subsets of papillary and medullary thyroid cancers (PTC and MTC), but targeted therapy for RET-driven cancers is currently clinically unavailable. Some mulit-kinase inhibitors, such as sunitinib and vandetanib, which have shown efficacy against other neoplastic diseases, are being evaluated in clinical trials for thyroid cancer therapy. However, the anti-VEGFR activities of these mulit-kinase inhibitors have been considered to lead cardiotoxicity in some patients. Moreover, Ret mutation-associated resistance have been reported by some RET inhibitors in preclinical data. The “in silico model” was applied to design the specific RET target compounds through mapping the pharmacophore of RET ATP-binding pocket. Some novel scaffold compounds which fit the pharmacophore model were selected form ITRI library, and their anti-RET activities were evaluated by kinase assay. The in vitro anti-cell proliferation activities of the compounds to the TT cells (MTC specific cells) were demonstrated by MTT assay. From the screening of ITRI library, ITRI-305 showed the impressive anti-RET (IC50 ∼ 90 nM) and anti-cell proliferation (IC50 at low micro molar) activities and the activities are compatible to those of sunitinib and vandetanib. ITRI-305 also shows good solubility and favorable oral PK profiles. The xenograft study of TT cells with ITRI-305 is proceeding. In conclusion, a novel scaffold RET inhibitor, ITRI-305, with less potent VEGFR activity (anti-VEFGR IC50 ∼ 1 μM) is developed to overcome the cardiotoxicity and resistance problems which is caused by conventional RET 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 728.
Abstract 729: Mechanism of action of a p53-derived peptide that targets a novel death pathway inducing selective necrosis in cancer cells
Authors: Bowne Wilbur B. , Yazdi Ehsan S. , Sookraj Kelley A. , Fenelus Maly , Adler Victor , Zenilman Michael E. , Michl Josef , Pincus Matthew R.
Published: February 2011

In a series of preliminary in vitro and in vivo studies we have shown that p53-derived peptides consisting of residues 12-26 and 17-26 from the murine double minute binding domain (MDM-2) possess anti-cancer activity. These peptides are linked to a trans-membrane penetrating sequence (penetratin) derived from the antennapedia homeodomain of Drosophila and are designated PNC-27 and PNC-28, respectively. We also found that the previously determined 3D structure of these PNC residues are directly superimposeable on the same residues bound to MDM-2, suggesting that these peptide may target MDM-2 in cancer cells. These peptide constructs appear to promote cancer cell death through a novel pathway that leads to necrosis rather than apoptosis while sparing their untransformed counterparts. In our initial experiments, the critical role of the MDM-2 oncoprotein as a potential target molecule for these PNC peptides was elucidated when a sequence specific MDM-2 antibody blocked PNC-27 activity on cancer cells. To further elucidate this anti-cancer mechanism we then tested parent molecule PNC-27 against selected cancer cell lines (Panc-02, pancreatic; HT1080, fibrosarcoma; and MCF-7, breast) plus respective untransformed counterparts (BMRPA1, BJ, and MCF-10) which were treated with PNC-27 and control peptide. Subsequently, treatment with fluorescent labeled peptide, immunoprecipitation (IP), confocal, and time-lapse electron microscopy (EM) was employed as well as transfection of untransformed cells with the MDM-2 gene to study the anti-cancer mechanism. PNC-27 did not cause elevation of pro-apoptotic proteins but induced rapid dose-dependent tumor cell death with release of lactate dehydrogenase (LDH). This specific anti-cancer mechanism, in part, may be due to observed increased expression of MDM-2 found in cancer cells compared to their normal cellular counterparts which is consistent with abrogation of MDM-2 and stabilization of p53 protein levels after PNC-27 treatment. Moreover, following IP of fluorescent labeled PNC-27 from treated cancer cells; labeled MDM-2 was detected. Furthermore, confocal microscopy and EM revealed co-localization of PNC-27 and MDM-2 along with cancer cell membrane pore formation, respectively. Remarkably, transfection of the MDM-2 gene resulted with increased expression of MDM-2 and now untransformed cell susceptibility to PNC-27. These results suggest a mechanism of PNC-27 binding to MDM-2 in the cancer cell membrane, leading to pore formation and membrane disruption; a p53-independent pathway consistent with rapid selective cancer cell necrosis.

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 729.
Abstract 731: 4-Amino-substituted 5-resorcinol-isoxazole derivatives, a novel class of Hsp90 inhibitor: Synthesis and evaluation of in vitro activity
Authors: Giannini Giuseppe , Cabri Walter , Brunetti Tiziana , Ciacci Andrea , Simoni Daniele , Baruchello Riccardo , Grisolia Giuseppina , Rullo Cinzia , Vesci Loredana , Castorina Massimo , Milazzo Ferdinando Maria , Carminati Paolo , Pisano Claudio
Published: February 2011

Hsp90 (heat shock protein 90) is a component of a molecular chaperone complex, involved in the folding, maturation and stabilisation of key signalling molecules which control cell proliferation, survival and transformation. It works by a modulation of a set of cancer-associated proteins, collectively referred to as “clients”. Inhibition of Hsp90 causes simultaneous destabilization and eventual degradation of client proteins that result in suppression of tumor growth. This observation led to the idea, among pharmaceutical chemists, to consider Hsp90 as a potential target for a new strategy in human cancer therapy. After pioneering studies with natural products (geldamycin and radicicol), many selective Hsp90 inhibitors from various institutions have entered in clinical trials. One of these classes of inhibitors is the 4,5-diaryl-isoxazole with a resorcinol in position 5 and a second substituted aryl in position 4. This scaffold was considered a prerequisite for the activity on Hsp90.

VER-52296/NVP-AUY922, currently in Phase II clinical trials, belongs to diaryl-isoxazole class.

In our project, we systematically investigated - with in silico pre-screening, in concert with structural information from X-ray protein crystallography - all possible modifications to make on the isoxazole scaffold. We have mainly focused our attention on the importance of an amide portion at the 4 position. Now we have identified a new class of synthetic small molecule Hsp90 inhibitors, characterized by a 4-amino-substituted 5-monoaryl-isoxazole (5-resorcinol-isoxazole) scaffold. We turned our attention to the 4 position of isoxazole where unexpectedly, we have found that compounds possessing a nitrogen atom directly attached to the isoxazole ring are endowed of activity against Hsp90 similar or even better than the diaryl analogues.

Here we describe design, synthesis and preliminary in vitro biological profile of a small library of these derivatives.

Examples from this series of compounds have activities ranging from low to high nM against Hsp90 as measured in a fluorescence polarization (FP) competitive binding assay, and are active in human cancer cell lines where they inhibit cell proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72.

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 731.
Abstract 733: Synthesis and biological evaluation of inhibitors of Rho protein kinase
Authors: Pireddu Roberta , Sun Nan N. , Yu Huidong , Sung Shen-Shu , Sebti Said M. , Lawrence Nicholas J.
Published: February 2011

Rho GTPases are small G-proteins which play a critical role in signaling pathways and control cell growth and division. Rho-associated protein kinase (known as ROCK or Rho kinase) is a Ser/Thr protein kinase activated by GTP-bound Rho and phosphorylates important signalling proteins. Rho/ROCK mediated signalling pathways are implicated in cell morphology, moltility, smooth muscle contraction, formation of stress fiber, focal adhesion, cell transformation and cytokineses. ROCKs have been subject to growing attention, having been implicated in a range of therapeutic areas including cancer. Further, the pharmacological inhibition of ROCKs has been suggested as a promising strategy in the prevention of cell invasion, a central event in the process of metastasis.

We describe the identification of potent ROCK inhibitors from data mining of the Molecular Libraries Screening Center Network (MLSCN) data available through PubChem and in-house HTS screening. Libraries of compounds have been designed to aid the hit-to-lead process and probe structure activity relationships. Molecular modeling and crystallography have been used to aid the design of potent ROCK inhibitors. Compounds were first assessed for in vitro ROCK1 inhibitory activity using a Z-lyte kinase assay. Selected compounds were also screened against the isoform ROCK2. Selectivity trends will be discussed. The overall process has led to the improvement of activity of compounds from high micromolar activity to low nanomolar activity. Structure activity relationship studies led to the identification of highly potent and cell-permeable analogues that were shown to inhibit selected substrates of ROCK such as MYPT1 in cell assays and are the subject of ongoing antitumor studies.

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 733.
Abstract 742: Development of novel naphthalimide derivatives as potential melanoma therapeutics
Authors: Sk Ugir Hossain , Gowda A.S. Prakasha , Gimbor Melissa A. , Gowda Raghavendra , Robertson Gavin P. , Spratt Thomas E. , Yun Jong K. , Amin Shantu , Sharma Arun K.
Published: February 2011

Over the years, several naphthalimide analogs with varying efficacy have been reported as having promising anti-cancer properties. Some analogs such as mitonafide, a tolemerase inhibitor, have shown an interesting antitumor activity both preclinically and in phase I and phase II clinical trials. However, it suffers from severe toxicity issues, especially central nervous system (CNS) toxicity. Because of toxicity issues, mitonafide could only be delivered by i.v. infusion, and still CNS toxicity is a major issue. This problem was overcome by adapting a delivery strategy of 5 days continuous infusion, but this regimen was not effective in NSCLC or colorectal cancer patients, while still having severe myelotoxicity in the latter case. These observations warranted optimization of mitonafide structure by adding appropriate functional groups that could help reduce systemic toxicity, while keeping the key naphthalimide moiety intact. We hypothesized that incorporation of functional groups such as isothiocyanate group which is present various naturally occurring agents found in cruciferous vegetables would enhance the bioavailability and reduce systemic toxicity levels. By analogy, isoselenocyanate group which has recently been shown by us to follow a similar mechanism with enhanced potency was also introduced. Furthermore, we also synthesized a series of mitonafide analogs with functional alterations such as thiourea and selenourea in place of N,N-dimethyl functionality in mitonafide, with or without nitro substitution. Interestingly, all the sulfur analogs [IC50 (72 h): 2-9 uM] were more cytotoxic than their corresponding selenium derivatives [IC50 (72 h): 11-24 uM], as observed by the MTS assay in various human melanoma cell lines. This was in sharp contrast to our earlier studies, where isosteric selenium analogs were more effective in killing multiple cancer cells and about 3 times more effective in inhibiting melanoma tumor development. Furthermore, the sulfur analogs effectively induced apoptosis in UACC 903 human melanoma cells. Nitro substitution or increase in alkyl chain length did not seem to make any significant difference in the activity of these compounds. Though the IC50 values were 2-4 times lower for mitonafide [IC50 (72 h): 1-2 uM] compared to novel isothiocyante or thiourea derivatives, these new derivatives were well tolerated when injected i.p. in mice everyday for a month. The new naphthalimide derivatives thus hold solid promise as melanoma therapeutics.

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 742.
Abstract 743: Assessment of novel tyrosine-chlorambucil hybrid molecules on a panel of breast, ovarian, and uterine cancer cell lines
Authors: Leblanc Valerie , Descoteaux Caroline , Parent Sophie , Berube Gervais , Asselin Eric
Published: February 2011

Site-specific drug targeting is the suject of very intense research to improve selectivity and therapeutic index towards breast and gynecological (ovarian and endometrial) cancers. Recently, our group have designed and synthesized a new class of amino acid-linked nitrogen mustard hybrids. The new hybrids are designed to mimic 17β-estradiol in order to target ERα-overexpressing tumor cells. For this purpose, tyrosine was used as the starting material to which chlorambucil was added to construct several tyrosine-chlorambucil hybrids. The phenol group of the tyrosine moiety imitates the phenol group of estradiol. Thus, the new hybrids are designed to target the estrogen receptor and to direct the cytotoxic moiety to hormone-dependent cancer cells. Chlorambucil is a slow acting nitrogen mustard mainly used in chronic lymphocytic leukemia and primary macroglobulinemia. It is also useful in treating lymphosarcoma and Hodgkin's disease. Two different families of hybrids were made. The aim of the study was to determine the cytotoxic effects of these unique molecules using estrogen dependent (ER+; Ishikawa, Ovcar-3, MCF-7, ZR-75-1) and independent (ER-; Hec-1A, A2780, MDA-MB-468) human breast, ovarian and uterine cancer cells. The results show the possible interactions of the two families of hybrids with ERα as demonstrated by molecular modeling. The biological activity of these compounds was evaluated in vitro using the MTT cell proliferation assay. Depending of the different hybrids tested the novel compounds generally possess up to 10 time higher biological activities when compared to the parent drug. Given the significant cytotoxic activity of these original tyrosine-chlorambucil hybrids, we believe that they represent a promising alternative treatment for hormone-dependent female cancers.

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 743.
Abstract 745: Structure-activity relationship study of the novel aspirin-based anticancer compounds
Authors: Joseph Stancy J. , Nie Ting , Johnson Francis , Gupta Ramesh , Rigas Basil
Published: February 2011

We performed a structure-activity relationship (SAR) study of novel derivatives of aspirin (ASA) which show strong anticancer activity in vitro and in vivo. We synthesized a series of ASA-based benzyl esters (ABEs) and evaluated their inhibitory activity against human colon (HT-29 and SW480) and pancreatic cancer cell lines (BxPC-3 and MIA PaCa-2). The ABEs that we studied largely comprise organic benzyl esters bearing an ASA or acyloxy group (X) at the meta or para position of the benzyl ring and one of four different leaving groups. The nature of the salicyloyl/acyloxy function, the leaving group, and the additional substituents affecting the electron density of the benzyl ring, all were influential determinants of the cancer cell growth inhibitory activity of each ABE. Positional isomerism also played a significant role in this effect. Our SAR study provides an insight into the biological properties of this novel class of compounds and underscores their potential as anticancer 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 745.
Abstract 749: Predicting and improving potential anticancer proteomimetics with computational modeling
Authors: Chen Kevin
Published: February 2011

In this work, computational approaches were used to investigate peptides for possible anticancer drug applications. Programmed cell death (apoptosis) is an important cellular process. Its malfunction can lead to cancer and other diseases. Apoptosis is regulated by both pro-death and pro-survival proteins. An over expression of pro-survival proteins can trigger cancer progression and poor chemotherapy response. A class of recently designed proteo-mimetics may help trigger death in cancer cells by hindering expression of certain pro-survival proteins.

The Deep View program and the Swiss Model server were used to model interactions between pro-survival Bcl-xl protein and pro-death Bim protein. First, all binding residues of the Bcl-xl-Bim complex were identified. Bim residues were then systematically mutated. The goal was to study the significance of side chain groups for the complex formation. The changes in binding free energy, caused by the mutations, were calculated.

Most mutations had a negligible influence on Bcl-xl-Bim interactions.

However, some mutations produced additional bonds with Bcl-xl. Several residue substitutions led to minimization of free energies. Most of these residues were binding either at the original Bcl-xl binding or in its vicinity. The obtained minimum-energy peptides suggested putative proteomimetics that could form strong bonds with Bcl-xl. These could cause Bim to dislodge from the Bcl-xl binding site and thus stimulate apoptosis. The potential of the mutated peptides to hinder tumor cells’ survival is discussed.

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 749.
Abstract 751: Synthesis of irofulven analogs
Authors: Kelner Michael J. , McMorris Trevor C.
Published: February 2011

Irofulven (NSC 683863), a semi-synthetic analog of the fungal product illudin S, that has been evaluated in a variety of phase I and II clinical trials. Promising results were noted in clinical trials against a variety of solid cancers including colon, sarcoma, prostate, liver, ovarian and pancreas. Irofulven in a randomized phase IIB trial increased median overall lifespan in patients with metastatic taxane-refractory prostate cancer [HR = 0.69]. As irofulven was the first member of the acylfulvene class of agents to be created, we synthesized several hundred analogs for further testing. Modifications were specifically made to 7 sites of the irofulven molecule including the cyclopropane ring, the chiral center, the carbonyl group, the tertiary hydroxyl site, and several nucleophilic sites. Modifications at several sites completely eliminated both the cytotoxicity and antitumor activity of the analog. Analogs with modifications at one site retained cytotoxic activity, as well as in vivo toxicity in rodents, but surprisingly eliminated antitumor activity in xenograft models. Analogs in which the tertiary hydroxyl group of irofulven was replaced with specific functional groups were noted to have an increase in tumor selectivity as noted by both cell culture and xenograft studies. Specific functional groups noted to be favorable for increased activity included carbamate, sulfonamide, and urea moieties. Additional derivatives of each of these specific functional groups were synthesized and tested in a variety of cell culture and xenograft studies including multidrug resistant models. These studies identify molecular sites critical for the antitumor activity of acylfulvenes, as well as identify promising new analogs for further investigation.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 751.
Abstract 758: Development of novel anticancer agents based on fusarochromanone
Authors: Mahdavian Elahe , Buckley Bonnie , Sermons Matthew , Clifford John L. , Furmanski Brian D. , Salvatore Brian A.
Published: February 2011

Cancer is the leading disease-related killer in the US, and therefore there is tremendous need for effective new chemotherapeutic treatments. The ultimate goal of this research project is to discover and develop novel chemotherapeutic agents of more potency and less toxicity for treatment of cancer patients. Increasingly, researchers are attempting to optimize bioactive natural products in order to enhance their efficacy and minimize their toxicity. The primary goal of this research is to develop novel anti-cancer agents based on fusarochromanone (FC101a), a natural mycotoxin with potent anti-angiogenic and direct anti- tumor activity. Like most other bioactive natural compounds, the potency of FC101a is compromised in-vivo, suggesting rapid metabolism and/or poor bioavailability and distribution. Clearly, further development of FC101a as a chemotherapeutic drug requires that we complete its total synthesis, as well as the synthesis of a series of structural analogs that may show greater in-vivo potency. The creation of the parent FC101a and its analogs will allow for the analysis of quantitative structure/activity relationships (QSAR), using both in-vitro cellular and in-vivo animal models of tumorigenesis.

Our research group has already synthesized a series of simplified FC101 analogs without the side chain, and we have conducted preliminary assays to determine the effect of these analogs on in-vitro cancer-cell viability and proliferation. In contrast to the parent compound, most of these new synthetic analogs that lack the side chain found in the natural product have increased cell viability and proliferation for all cell lines tested. This sharp contrast in activity indicates that the FC101 side chain is required for the anti-cancer and angiogenic activity of this molecule. Although the stimulation of proliferation may have profound applications (e.g., the stimulation of stem cell growth), we are focusing our current research efforts on the natural product itself as well as new side chain analogs that may find direct application in the treatment of 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 758.
Abstract 760: Sensitizing tumors to chemotherapy by inhibition of gamma-glutamyl transpeptidase
Authors: LaChapelle Stephanie , Cook Paul F. , Hanigan Marie H.
Published: February 2011

Tumors that express the cell surface enzyme gamma-glutamyl transpeptidase (GGT) have increased resistance to chemotherapy and radiation. GGT cleaves the gamma-glutamyl bond of extracellular glutathione, initiating the cleavage of glutathione into glutamate, cysteine and glycine. Glutathione can not be taken up intact by tumor cells. Cleavage of extracellular glutathione increases the supply of cysteine to the cell, which is essential to increasing intracellular glutathione levels. Glutathione detoxifies chemotherapy drugs, and elevated levels of intracellular glutathione can block apoptosis. We are developing inhibitors of GGT that can be used to sensitize tumors to therapy. The standard assay for GGT activity uses p-nitroanilide derivatives as substrates, millimolar concentrations of dipeptides as acceptors and is buffered at pH 8.6. Under these assay conditions several groups of investigators have shown that GGT cleaves the gamma-glutamyl bond of the substrate and transfers the gamma-glutamyl group to an acceptor by a Ping-Pong mechanism. However, the standard assay for GGT activity does not measure the reaction catalyzed by GGT in vivo. We have assayed GGT activity under physiologic conditions. We have found that some compounds that inhibit GGT activity in the standard reaction do not inhibit under physiologic conditions. Our data reveal that, at physiologic pH and osmolarity with reduced concentrations of acceptor, the enzyme does cleave the gamma-glutamyl bond by a Ping-Pong mechanism, but hydrolysis of the intermediate of the gamma-glutamyl enzyme intermediate is significant, and competition between water and the peptide acceptor for the intermediate makes it appear Sequential. Several compounds that inhibit GGT in the standard assay actually accelerate activity as the concentration of the acceptor is reduced to physiologic levels. To identify inhibitors that are optimized for clinical use, we have developed an assay that evaluates GGT cleavage of glutathione, the physiologic substrate, at physiologic pH and osmolarity. We are using our previously reported inhibitor OU749 and other lead compounds to further characterize the physiologic GGT reaction and to identify essential substructural components for inhibition of GGT activity.

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 760.
Abstract 761: The development of cell permeable Shp2 PTP inhibitors
Authors: Lawrence Harshani R. , Chen Liwei , Pernazza Daniele , Scott Latanya , Ren Yaun , Luo Yunting , Wu Xin , Shen-Shu Sung , Guida Wayne , Sebti Said M. , Lawrence Nicholas J. , Wu Jie
Published: February 2011

Protein tyrosine phosphorylation affects cellular activities that control tumor growth and progression. Protein tyrosine phosphorylation, a key regulatory process of signal transduction pathways, is controlled by the action protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Kinases are now a clinically proven anticancer drug target. The development of PTP inhibitors is, by comparison, a relatively new field. Since PTPs do not require a small molecule cofactor they generally show a low propensity to bind a small molecule inhibitor. Nevertheless there are now examples of highly potent PTP inhibitors that provide promising proof-of-principle that PTPs are druggable. Most PTP inhibitors have a charged or highly polar functional group that mimics the phosphotyrosine residue, that often leads to poor ADME and cell permeability properties.

We will report our efforts to address the problems of cell permeability we have encountered in our Shp2 PTP inhibitor program. We have previously reported a series of isatin based Shp2 inhibitors based on a compound (NSC-117199) identified from screen of the NCI Diversity Set. We synthesized >100 analogs of NSC-117199 in our lead optimization effort, which yielded several compounds with approximately 50-fold increase in potency and > 10-fold increase in selectivity (Shp2 versus Shp1). However, these Shp2 PTP inhibitors have either a polar nitro or carboxyl group and have no detectable cellular activity. We will report three approaches to address this problem. The first approach was to prepare neutral ester prodrugs. Several compounds have clearly demonstrable activity against Shp2 in cells. A series of esters has also been designed to also improve the water solubility of the PTP inhibitor. A second approach taken was to further modify the isatin scaffold that does not necessitate the use of a prodrug strategy. We will report the discovery of new isatins related to NSC-117199 that have new modifications that result in cell active Shp2 inhibitors. Finally we will report the discovery and preliminary optimization of new classes of cell active Shp2 inhibitors based on alternative scaffolds.

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 761.