bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4445: Dual ALK and EGFR inhibition targets a mechanism of acquired resistance to the tyrosine kinase inhibitor crizotinib in ALK translocated lung cancer.
Authors: Yamaguchi Norihiro , Nakayama Sohei , de Figueiredo-Pontes Lorena L. , Kobayashi Susumu , Costa Daniel B.
Published: August 2014

ABSTRACT

Introduction: The multitargeted tyrosine kinase inhibitor (TKI) crizotinib is active against ALK translocated non-small-cell lung cancer (NSCLC); however acquired resistance invariably develops over time. ALK mutations have previously been implicated in only a third of resistant tumors. We sought to evaluate alternative mechanisms of resistance and preclinical strategies to overcome these in a cell line driven by EML4-ALK.

Methods: We selected the NSCLC cell line NCI-H3122 (H3122: EML4-ALK E13;A20) and derived resistant variants that were able to grow in the presence of 1μM crizotinib. These were analyzed for ALK mutations, sensitivity to crizotinib in combination to other TKIs (imatinib, sorafenib, erlotinib and afatinib), and for activation of alternative tyrosine kinases.

Results: All H3122 crizotinib resistant (CR) clones lacked mutations in the kinase domain of ALK. To evaluate if possible alternative kinases functioned as “bypass” tracks for downstream signaling activation in these resistance cells, we used a functional approach of dual ALK TKI (with crizotinib) with combinatory TKI inhibition to screen for possible targets. Crizotinib + sorafenib (BRAF, RET, PDGFR, VEGFR TKI) and crizotinib + imatinib (ABL, PDGFR, KIT TKI) were ineffective. In contrast, crizotinib + erlotinib (reversible EGFR TKI) and crizotinib + afatinib (irreversible EGFR/ERBB2 TKI) were able to inhibit the growth of H3122 CR clones, indicating potential EGFR activation as a mechanism of resistance. Analyses of phosho-receptor tyrosine kinase arrays demonstrated that CR clones had 5-25 times higher phospho-EGFR signal than H3122 parental cells did after the exposure to 1μM crizotinib, indicating phospho-EGFR was the most predominant phosphorylated signal in crizotinib-resistant cells.

Conclusions: Using a functional approach of combining ALK and EGFR TKIs, we identified activation of EGFR as a mechanism of resistance to crizotinib in preclinical models of ALK translocated NSCLC. If EGFR activation is confirmed as a predominant mechanism of crizotinib resistance in patient-derived tumors, the use of crizotinib + EGFR TKIs should be tested in future clinical trials for this important cohort of NSCL

Citation Format: Norihiro Yamaguchi, Sohei Nakayama, Lorena L. de Figueiredo-Pontes, Susumu Kobayashi, Daniel B. Costa. Dual ALK and EGFR inhibition targets a mechanism of acquired resistance to the tyrosine kinase inhibitor crizotinib in ALK translocated lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4445. doi:10.1158/1538-7445.AM2013-4445
Abstract 4456: Biochemical and structural characteristics of crizotinib-resistant ALK mutants.
Authors: Timofeevski Sergei , Liu Wei , Deng Ya-Li , Brooun Alexei , Bergqvist Simon , Karlicek Shannon , Murray Brion , Bolaños Ben , McTigue Michele
Published: August 2014

Anaplastic lymphoma kinase (ALK), when aberrantly regulated through gene translocations or mutations, has been implicated as an oncogenic driver in a variety of cancers. XALKORI (crizotinib) is an FDA-approved therapy for locally advanced or metastatic (ALK)-positive non-small cell lung cancer (NSCLC). Mutations of certain residues within the ALK kinase domain have been associated with crizotinib resistance. To understand the molecular basis of mutational resistance, we have kinetically and structurally characterized several clinical mutations of ALK kinase domain. In the basal-state (nonphosphorylated) proteins, mutations C1156Y, F1174L, L1152R and L1196M resulted in 14 to 52-fold increase in catalytic efficiency of phosphorylation of an activation loop peptide. Accordingly, these mutants were more rapidly autophosphorylated, compared to wild-type enzyme. In addition, F1174L, C1156Y and L1156Y had 2 to 6-fold higher catalytic efficiencies when fully activated by autophosphorylation. Conversely, catalytic efficiencies of nonphosphorylated and phosphorylated G1269A variant decreased by 1.2 and 1.6-fold, respectively. Inhibition of L1152R, L1196M and G1269A by crizotinib was reduced by 3-28-fold, compared to wild-type enzyme, from Ki determinations. From direct binding studies of the L1196M “gatekeeper” mutation, the loss of crizotinib binding occurred 6 and 13-fold for phosphorylated and nonphosphorylated proteins, respectively. By crystallographic studies apoenzyme and crizotinib complexes of ALK kinase domain displayed an inactive kinase conformation that is stabilized by an extended hydrophobic network of residues. F1174 and L1196 are part of this hydrophobic core, and mutations of these residues are predicted to destabilize the inactive ALK conformation. Taken together, these results suggest that most of the resistant mutations (e.g. L1196M, F1174L, C1156Y, L1152R) result in a more dynamic protein that increases substrate turnover. In addition, the reduction of crizotinib binding for the mutations in the vicinity of the inhibitor binding site (L1196M, G1269A) is likely a contributing factor for the resistance. This structural and kinetic analysis of mutational resistance may be useful for design of new inhibitors targeting multiple clinical mutations of ALK.

Citation Format: Sergei Timofeevski, Wei Liu, Ya-Li Deng, Alexei Brooun, Simon Bergqvist, Shannon Karlicek, Brion Murray, Ben Bolaños, Michele McTigue. Biochemical and structural characteristics of crizotinib-resistant ALK mutants. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4456. doi:10.1158/1538-7445.AM2013-4456
Abstract 4468: Cripto-1 elicits innate resistance to EGFR inhibitors in non-small cell lung cancer harboring EGFR-sensitizing mutations.
Authors: Park Kang-Seo , Giuseppe Giaccone , Wang Yisong , Pham Trung , Moon Yongwha , Luo Ji , Lee Liam Changwoo , Mitsudomi Tetsuya , Okamoto Isamu , Yatabe Yasushi , Mok Tony , Raffeld Mark , Xi Liqiang , Salomon David S. , Bianco Caterina
Published: August 2014

Non-small cell lung cancer (NSCLC) carrying sensitizing epidermal growth factor receptor (EGFR) mutations can initially benefit from erlotinib (EGFR inhibitor) treatment. However, patients invariably develop resistance through secondary EGFR mutations, c-MET amplification, epithelial-mesenchymal transition (EMT), small cell lung cancer transformation, PIK3CA mutation and other unknown mechanisms. Here we examined whether Cripto1, which has been shown to induce EMT, could cause erlotinib resistance. We show that innate erlotinib-resistant lung adenocarcinomas carrying sensitizing EGFR mutations exhibit higher level of Cripto1 expression than their erlotinib sensitive counterparts. Ectopic expression of Cripto1 rendered erlotinib-sensitive lung adenocarcinoma cells resistant to erlotinib both in vitro and in a xenograft model. Moreover, Cripto1 activated Zeb1/EMT signaling pathway in EGFR-TKI resistant cells. High Cripto1 expression correlated with poor prognosis of NSCLC patients. Our study provides a novel Cripto1-dependent mechanism of EGFR-TKI resistance that may pave the way for the development of therapies to overcome EGFR-TKI resistance in lung cancer patients.

Citation Format: Kang-Seo Park, Giaccone Giuseppe, Yisong Wang, Trung Pham, Yongwha Moon, Ji Luo, Liam Changwoo Lee, Tetsuya Mitsudomi, Isamu Okamoto, Yasushi Yatabe, Tony Mok, Mark Raffeld, Liqiang Xi, David S. Salomon, Caterina Bianco. Cripto-1 elicits innate resistance to EGFR inhibitors in non-small cell lung cancer harboring EGFR-sensitizing mutations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4468. doi:10.1158/1538-7445.AM2013-4468
Abstract 4482: Pemetrexed treatment results in DNA replication fork instability and double strand breaks formation in UNG-/- human cancer cells.
Authors: Weeks Lachelle D. , Zentner Gabriel , Scacheri Peter , Gerson Stanton L.
Published: August 2014

Misincorporation of genomic uracil and formation of DNA double strand breaks are known consequences of exposure to TS inhibitors such as 5-fluorouracil, and pemetrexed. Uracil DNA glycosylase catalyzes the excision of genomic uracil and initiates DNA base excision repair (BER). Thus, a relationship between antifolate cytotoxicity and UNG expression and activity has been hypothesized. However, a precise mechanism linking antifolate-induced formation of DSBs to genomic uracil accumulation and UNG-initiated BER has not been described. Herein, we report that despite equivalent proliferation indices, DLD1 UNG-/- cells are more sensitive to pemetrexed mediated intra S-phase arrest, DNA double strand break formation and apoptosis compared to UNG+/+ cells. Using data from western blots in chromatin extracts, PCNA staining of cells in S-phase, and pulse-chase labeling of replicating cells with CldU and IdU, we surmise that the accumulation of uracil in pemetrexed-treated UNG-/- cells is associated with significant replication fork instability. In addition, UNG-/- cells have reduced capacity to recover from pemetrexed-mediated DNA damage, as indicated by the persistence of S-phase arrest and gamma-H2AX foci. This defect in recovery was not explained by double strand break repair capacity, which was equivalent in UNG+/+ and UNG-/- cells. Using γ-H2AX ChIP sequencing, we observed a 5-fold increase in the number of γ-H2AX binding sites in UNG-/- cells compared to UNG+/+ cells treated at IC50 levels of pemetrexed. This analysis evinced distinct patterns of γ-H2AX binding in UNG+/+ and UNG-/- cells. Double strand breaks (γ-H2AX) were more significantly associated with transcription start sites and putative origins of replication in UNG-/- cells compared to UNG+/+ cells. Based on these data we conclude that uracil accumulation, and thus UNG activity, during pemetrexed exposure directs both the quantity and the location of double strand breaks. These findings support uracil mediated S-phase arrest and DNA replication fork collapse as the mechanism of double strand break formation and cell death in pemetrexed treated UNG-/- cells.

Citation Format: Lachelle D. Weeks, Gabriel Zentner, Peter Scacheri, Stanton L. Gerson. Pemetrexed treatment results in DNA replication fork instability and double strand breaks formation in UNG-/- human cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4482. doi:10.1158/1538-7445.AM2013-4482
Abstract 4484: Uracil DNA glycosylase expression determines human lung cancer cell sensitivity to pemetrexed.
Authors: Weeks Lachelle D. , Fu Pingfu , Liu Lili , Gerson Stanton L.
Published: August 2014

Uracil misincorporation into DNA is a consequence of pemetrexed inhibition of thymidylate synthetase (TS). The DNA base excision repair (BER) enzyme, uracil DNA glycosylase (UNG) is the major glycosylase responsible for removal of misincorporated uracil. We previously illustrated hypersensitivity to pemetrexed in UNG-/- human colon cancer cells (DLD1) and proposed that cytotoxic uracil accumulation leads to collapsed replication forks, double strand breaks and cell death. Here, we examined the relationship between cellular capability to remove uracil-DNA and pemetrexed sensitivity in human lung cancer cells. Viral siRNA and shRNA knockdown of UNG enhanced pemetrexed sensitivity in A549 and H1975 cells, respectively. We further evaluated induction of UNG as a mechanism of pemetrexed resistance. Exposure of H1299 cells to step-wise increasing concentrations of pemetrexed established drug resistant sublines: H1299PR-1 and H1299PR-2. Significant induction of UNG protein was observed in both sublines. Importantly, co-treatment with the BER inhibitor, methoxyamine (MX), overcame pemetrexed resistance in H1299PR-1/2 underscoring the utility of BER directed therapeutics to offset acquired pemetrexed resistance. Lastly, differential expression of UNG in cell lines and primary lung cancer tissues was associated with pemetrexed sensitivity. Cell lines derived from squamous and small cell carcinoma, known to be clinically resistant to pemetrexed, had the highest levels of UNG expression in a panel of eight lung cancer cell lines. Analysis of cDNA microarrays derived from primary human lung cancer tissues also indicated high UNG expression in pemetrexed-resistant histological subtypes. Thus, we propose that UNG is a major determinant of pemetrexed sensitivity in human lung cancer.

Citation Format: Lachelle D. Weeks, Pingfu Fu, Lili Liu, Stanton L. Gerson. Uracil DNA glycosylase expression determines human lung cancer cell sensitivity to pemetrexed. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4484. doi:10.1158/1538-7445.AM2013-4484
Abstract 4487: Synergistic effects of resveratrol and doxorubicin on inducing the apotosis of mouse and human PTEN deficient T-cell acute lymphoblastic cells.
Authors: Chen Ling , Yang Yaling , You M. James
Published: August 2014

Resveratrol (3,5,4’-trihydroxy-trans-stilbene), a plant antibiotic produced in a wide variety of plants in response to stress, injury, UV irradiation, and fungal infection, has been shown to inhibit cell growth of several types of cancer. The aim of the present study was to determine the effects of Resveratrol alone and its combination with Doxorubicin, a commonly used chemotherapeutics for T-cell acute lymphoblastic leukemia (T-ALL), on the proliferation and apoptosis of T-ALL cells. Mouse T-ALL cell lines were derived from T-cell-specific deletion of Pten tumor suppressor gene, and human T-ALL cells were obtained from ATCC. We treated T-ALL cells with different concentrations of Resveratrol (0 μM to 200 μM) alone and in combination with various concentrations of Doxorubicin (0 μM to 1 μM) for different durations (0, 12, 24, 48 and 72 hours, respectively). We showed mouse and human Pten deficient T-ALL cell lines were sensitive to the treatment of Resveratrol in dose- and time-dependent manners. In addition, low dose Resveratrol (3.125 μM) in combination with very low dose of o (0.01 μM) showed a remarkable synergistic effect on the cell proliferation and apoptosis. Flow cytometric analysis with Annexin V and Western blot studies on Caspase 3 confirmed that Resveratrol induced apoptosis, especially in combination with Doxorubicin. This study establishes a potential role of Resveratrol in the treatment of Pten deficient T-ALL.

Citation Format: Ling Chen, Yaling Yang, M. James You. Synergistic effects of resveratrol and doxorubicin on inducing the apotosis of mouse and human PTEN deficient T-cell acute lymphoblastic cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4487. doi:10.1158/1538-7445.AM2013-4487
Abstract 4488: A synergistic effect between aldehydes and doxorubicin in S. cerevisiae provides a model for enhancing anthracycline-based therapy.
Authors: Miles Jana S.
Published: August 2014

We have previously reported that interruption of the synthesis of the amino acid threonine confers sensitivity to doxorubicin in S. cerevisiae. Threonine synthesis requires the genes HOM6, THR1, and THR4, which encode the enzymes homoserine dehydrogenase, homoserine kinase, and threonine synthase, respectively. The inactivation of these genes in threonine biosynthesis results in increased toxicity of doxorubicin. Interestingly, the most sensitive strain, hom6, results in the accumulation of L-aspartate semialdehyde. We hypothesize that the accumulation of this metabolite results in toxic synergy with doxorubicin.

We have further investigated the role of aldehydes in the sensitization of yeast cells to anthracyclines. Inactivation of HOM3, which encodes for the aspartate kinase required for the first step in threonine biosynthesis, prevents the accumulation of the L-aspartate semialdehyde in the hom6 strain and rescues its sensitivity to doxorubicin, suggesting a role for the aldehyde in the sensitization to doxorubicin. Furthermore, wild type strains treated with different aldehydes significantly reduced the survival of the cells to anthracyclines, and distinctly in other therapeutic agents. These results indicate that aldehydes have a synergistic effect with anthracyclines. In humans, threonine is an essential amino acid and its biosynthesis does not occur. However, our results are directly applicable to relevant human pathways in nonessential amino acids and ornithine synthesis. Potential application of the accumulation of glutamic semialdehyde and succinic semialdehyde intermediates for a synergistic effect with doxorubicin is discussed.

Research supported by NIH/NCRR RCMI grant #RR03020 at Florida A&M University.

Citation Format: Jana S. Miles. A synergistic effect between aldehydes and doxorubicin in S. cerevisiae provides a model for enhancing anthracycline-based therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4488. doi:10.1158/1538-7445.AM2013-4488
Abstract 4496: Differential inhibition by microtubule stabilizing agents of 2-(m-azidobenzoyl)Taxol photoaffinity labeling of tubulins from different eukaryotic sources.
Authors: Yang Chia-Ping H. , Xiao Hui , Horwitz Susan Band
Published: August 2014

Distinct α- and β-tubulin isotypes are present in different quantities in diverse eukaryotic cells. The tritium-labeled photoaffinity Taxol analog, 2-(m-azidobenzoyl)Taxol, was used to photolabel tubulins prepared from HeLa cells, bovine brain and chicken erythrocytes. A 5-fold molar excess of unlabeled compound inhibited the photolabeling of tubulin by >90%, demonstrating the specificity of this photolabeling. Tubulin isotype content varies from one source to another, particularly for chicken erythrocytes that contain only one α-tubulin and one β-tubulin (α1 and βVI). 2-(m-azidobenzoyl)Taxol photoaffinity labeling of tubulin was carried out in the presence of six different microtubule stabilizing agents, including Taxol, epothilone B, discodermolide, ixabepilone, laulimalide and peloruside A. The inhibitory effects elicited by Taxol, epothilone B, discodermolide and ixabepilone, at a 5-fold molar excess, on photolabeling are different for tubulins prepared from different sources. Taxol had a minimal inhibitory effect on HeLa and bovine brain tubulin, but a strong inhibitory effect on chicken erythrocyte tubulin. Discodermolide exhibited opposite effects on photolabeling, compared to those of Taxol. This is most likely due to the presence of distinct tubulin isotypes in different sources. However, laulimalide and peloruside A that are known to bind to a different site than Taxol in β-tubulin, stimulated photolabeling of tubulins from all three sources. Stathmin (S) plays an important role in the regulation of microtubule dynamics through sequestering tubulin (T) into a T2S complex. When stathmin was incubated, in the presence of 1 mM CaCl2, with chicken erythrocyte tubulin at a tubulin:stathmin ratio of 2:1, followed by incubation with tritium-labeled 2-(m-azidobenzoyl)Taxol in a buffer containing EGTA, photolabeling of β-tubulin was markedly reduced, suggesting that stathmin inhibited binding of Taxol to tubulin. Radiolabeled photoaffinity drug analogs are useful tools for determining drug binding affinities for the different tubulin isotypes. We are currently analyzing binding affinities of 2-(m-azidobenzoyl)Taxol to different bovine brain tubulin isotypes resolved by isoelectrofocusing.

Citation Format: Chia-Ping H. Yang, Hui Xiao, Susan Band Horwitz. Differential inhibition by microtubule stabilizing agents of 2-(m-azidobenzoyl)Taxol photoaffinity labeling of tubulins from different eukaryotic sources. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4496. doi:10.1158/1538-7445.AM2013-4496
Abstract 4505: Preparation and anti-tumor growth of polyethylene glycol-poly lactic acid block copolymers sorafenib nanoparticles.
Authors: Qin Jianmin
Published: August 2014

BACKGROUND&AIMS: In vitro and in vivo studies have showed that sorafenib can inhibit the growth of liver cancer cells and solid tumor. However, poor water solubility, short half-life, high side-effect limit its clinical application in malignant tumor treatment. In this study, we report on the preparation of sorafenib nanoparticles by nanotechnology, and to observe the anti-hepatocarcinoma effect and in vivo metabolism of sorafenib nanoparticles. Methods:Polyethylene glycol-poly lactic acid block copolymers were synthesized with ring-opening polymerization technique as drug carrier material. Polyethylene glycol-poly lactic acid block copolymers sorafenib nanoparticles were prepared with nanometer precipitation method. The particle diameter and surface potential of sorafenib nanoparticles were determined by nanometer particle size and zeta otentiometer(Nano-ZS ZEN3600, Malvern). Drug-loading rate and entrapment rate were determined by ultraviolet spectrophotometer. In vitro slow release of sorafenib nanoparticles was determined by extracorporeal dialysis. The effect of sorafenib nanoparticles on H22 liver cancer cells proliferation was determined with CCK8 reagent in vitro culture .In vivo pharmacokinetics of sorafenib nanoparticles was analysed with DAS2.1.1 software. Results:Sorafenib nanoparticles were round, particle diameter was 127.3±2.0 nm, Zeta potential was -3.35±0.42 mV, drug-loading rate was 6.78%, entrapment rate was 98.21%. In vitro accumulative release rate of sorafenib nanoparticles at 24h, 48h, 72h, 96h were respectively 50.9073%, 56.2395%, 60.2561%, 63.2843%. The inhibitive effect of sorafenib nanoparticles on H22 liver cancer cells proliferation was more enhanced than that of sorafenib at the same drug concentration(F=74.988,P<0.05), The inhibitive effect of sorafenib nanoparticles on H22 liver cancer cells proliferation enhanced at the increase of sorafenib concentration. The in vivo metabolism time of sorafenib in sorafenib nanoparticles group was longer than that of sorafenib group (SNP t1/2, 13.95h vs S t1/2, 9.67h; t=2.618, P<0.05). Conclusion:Sorafenib nanoparticles can significantly inhibit the cells proliferation of liver cancer, and prolong in vivo metabolism of srafenib. The preparation of sorafenib nanoparticles has established good theoretical basis to further study in vivo anti-tumor effect and mechanism of sorafenib nanoparticles .

Citation Format: Jianmin Qin. Preparation and anti-tumor growth of polyethylene glycol-poly lactic acid block copolymers sorafenib nanoparticles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4505. doi:10.1158/1538-7445.AM2013-4505
Abstract 4507: Utilizing the targeting properties of elastin-like polypeptide to safely deliver a potent doxorubicin derivative.
Authors: Walker Leslie R. , Perkins Eddie , Abu Ajaj Khalid , Kratz Felix , Raucher Drazen
Published: August 2014

N-butyldiacetate doxorubicin (NBD) is a derivative of a common chemotherapeutic agent that is three to four orders of magnitude more potent that the its parent compound. Targeting such a potent drug to the tumor, relies on an effective tumor targeting strategy. For this purpose, we have developed a hypothermic approach and developed a new macromolecular conjugate by coupling NBD to elastin-like polypeptide (ELP), a macromolecule that is soluble at normal physiological temperature but aggregates in the presence of heat (40-42 °C). Attaching NBD to ELP holds the promise of accumulating NBD effectively in the tumor while sparing healthy tissue. ELP is a macromolecule that is soluble at normal physiological temperature but aggregates in the presence of heat (40-42 °C). The ELP delivery system, SynB1-ELP-NBD, consists of 3 parts: a) a cell-penetrating peptide to facilitate entry through the cell membrane, b) ELP to allow thermal targeting, and c) an acid-sensitive derivative of doxorubicin, NBD. Preliminary results in the S2013 pancreatic cancer cell line have been promising. The IC50 of free doxorubicin is ∼9 μM, while the IC50 of SynB1-ELP-NBD is 1 nM. Very little is known about the mechanism of NBD inside the cell. Using flow cytometry, we compared apoptosis, cell cycle arrest, and generation of reactive oxygen species of SynB1-ELP, SynB1-ELP-NBD, and free NBD. While SynB1-ELP had very little effect on the cell cycle, generation of ROS, and less than 10% apoptotic cells, SynB1-ELP-NBD and free NBD were more active during the S phase at the expense of G1, generated 6× more ROS compared to control cells, and 80% of cells were apoptotic or necrotic after 72 h treatment. In vitro, SynB1-ELP-NBD works very similarly to free NBD, but safe delivery is the goal. In a maximum tolerated dose study, the animals were able to tolerate 2.5 times more NBD when it was attached to ELP compared to free NBD. This delivery system makes it possible to deliver larger amounts of a highly potent doxorubicin derivative to tumor-bearing animals and a comparative in vivo study in a pancreatic cancer mouse model is ongoing.

Citation Format: Leslie R. Walker, Eddie Perkins, Khalid Abu Ajaj, Felix Kratz, Drazen Raucher. Utilizing the targeting properties of elastin-like polypeptide to safely deliver a potent doxorubicin derivative. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4507. doi:10.1158/1538-7445.AM2013-4507
Abstract 4509: Genetically encoded chimeric polypeptide nanoparticles for paclitaxel delivery to triple negative breast cancer.
Authors: Bhattacharyya Jayanta , McDaniel Jonathan , Chilkoti Ashutosh
Published: August 2014

Triple negative breast cancer (TNBC) represents an important clinical challenge, because TNBC, like other breast cancer subtypes is highly metastatic, but TNBC displays a shorter median time to relapse and death. Chemotherapy remains the mainstay of treatment of TNBC, as it gives a markedly higher response rate than endocrine therapy or other available targeted agents. Current treatment strategies for TNBC include treatment with anthracyclines, taxanes, and platinates. Like other drugs, paclitaxel shows a lack of selectivity towards tumor cells, sub-optimal pharmacokinetics and bio-distribution, leading to limited efficacy and significant systemic toxicity. Although AbraxaneTM (albumin bound paclitaxel) an improved formulation of paclitaxel compared to the original chremophor formulation has been used to treat TNBC, it also suffers from systemic toxicity such as sensory neuropathy, neutropenia, nausea, and anemia.

In an effort to overcome the limited efficacy and systemic toxicity of current paclitaxel formulations, we have designed a novel polypeptide drug delivery system where paclitaxel is covalently attached to a genetically engineered chimeric polypeptide (CP) through a pH sensitive linker such that covalent conjugation of multiple copies of Paclitaxel at one end of the CP spontaneously triggers the self-assembly of the drug-CP conjugate into sub 100 nm size nanoparticles that are ideal for targeting solid tumors through the enhanced permeability and retention (EPR) effect.We found that in a human triple negative breast cancer cell (MDA-MB-231), the CP-paclitaxel conjugate showed substantial cytotoxicity that was similar to free drug. CP-Paclitaxel nanoparticle treated mice with orthotopic MDA-MB-231 tumors showed significantly greater tumor regression as well as enhanced overall survival compared to free drug. These results are promising because they demonstrate the potential of rationally engineered nanoparticle drug carriers to greatly improve the efficacy of currently approved cytotoxic agents for the treatment of solid tumors. In future work, we will evaluate the efficacy of CP-Paclitaxel nanoparticles combined with a tyrosine kinase inhibitor that mimics current combination chemotherapy.

Citation Format: Jayanta Bhattacharyya, Jonathan McDaniel, Ashutosh Chilkoti. Genetically encoded chimeric polypeptide nanoparticles for paclitaxel delivery to triple negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4509. doi:10.1158/1538-7445.AM2013-4509
Abstract 4529: Self-assembled lipid-polymer hybrid nanoparticles for the sustained delivery of siRNA and the treatment of drug-resistant cancers.
Authors: Shi Jinjun , Xu Yingjie , Xu Xiaoyang , Votruba Alexander , Zhu Xi , Pridgen Eric , Xiao Zeyu , Langer Robert , Zetter Bruce R. , Farokhzad Omid C.
Published: August 2014

RNA interference (RNAi), which can selectively knockdown target genes, has shown great potential in the treatment of various diseases including cancer. Thus far, numerous nanoparticle (NP) platforms such as lipoplex and polyplex have been developed to facilitate the safe and effective delivery of small interfering RNA (siRNA), which represents a major hurdle for the clinical applications of RNAi. Nevertheless, these NP systems lack the sustained siRNA release property, and thus can only induce transient gene silencing due to the short lifetime of siRNA. Therefore, the delivery of siRNA using controlled-release NPs would be necessary for achieving sustained gene silencing.

Herein, we will present a novel NP platform for safe and effective siRNA delivery in a sustained manner, which can be developed through the self-assembly of biodegradable and biocompatible polymers and lipids. The lipid-polymer hybrid NPs show excellent in vitro knockdown efficacy at low doses of siRNA, and promising in vivo results for delivering siRNA to xenograft tumors. More importantly, these NPs can control the temporal release of siRNA, with the half-release time of ∼ 9 days, for sustained silencing of target gene expression. Results demonstrate that the luciferase expression is still less than 25% at day 7 when the lucifrease-expressed HeLa cells were transfected with the NPs containing 40 pmol anti-luciferase siRNA. As a comparison, the luciferase expression is largely recovered (∼ 80%) at day 7, after transfection with lipo2000-siRNA complexes. Furthermore, this NP platform has been applied to deliver anti-drug resistance siRNA (e.g., anti-PHB1) and chemotherapeutic drugs (e.g., taxanes) for the effective treatment of drug resistant cancers. We believe that the lipid-polymer hybrid NP platform with the property of sustained siRNA release could hold potential in both fundamental studies and clinical applications.

Citation Format: Jinjun Shi, Yingjie Xu, Xiaoyang Xu, Alexander Votruba, Xi Zhu, Eric Pridgen, Zeyu Xiao, Robert Langer, Bruce R. Zetter, Omid C. Farokhzad. Self-assembled lipid-polymer hybrid nanoparticles for the sustained delivery of siRNA and the treatment of drug-resistant cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4529. doi:10.1158/1538-7445.AM2013-4529
Abstract 453: Immune cell profile of sentinel lymph nodes in melanoma patients .
Authors: Mohos Anita , Sebestyen Timea , Liszkay Gabriella , Plotar Vanda , Horvath Szabolcs , Gaudi Istvan , Ladanyi Andrea
Published: August 2014

Besides being a preferential site of early metastasis, the sentinel lymph node (SLN) is also a privileged site of T-cell priming, and may thus be an appropriate target for investigating cell types involved in antitumor immune reactions. We determined the prevalence of OX40+ activated T lymphocytes, FOXP3+ regulatory T cells, DC-LAMP+ mature dendritic cells (DCs) and CD123+ plasmacytoid DCs by immunohistochemistry in 100 SLNs from 60 melanoma patients. Density values of each cell type in SLNs were compared to those in non-sentinel nodes obtained from block dissections (n=37), and analyzed with regard to associations with clinicopathological parameters and disease outcome. Sentinel nodes showed elevated amount of all cell types studied in comparison to non-sentinel nodes. Metastatic SLNs had higher density of OX40+ lymphocytes compared to tumor-negative nodes, while no significant difference was observed in the case of the other cell types studied. Within the sentinel-positive group, FOXP3+ cell density was higher in patients showing disease progression and >5 years survival than in those without progression and <5 years survival, respectively, or in sentinel-negative cases. High amount of FOXP3+ cells in SLNs was associated with shorter progression-free (P=0.0203) and overall survival (P=0.0238), while no significant correlation was found in the case of the other immune cell types. In multivariate analysis, SLN status, ulceration, Breslow index and patient gender, but not the density values of the immune cell types, proved significant independent predictors of survival. Taken together, our results are compatible with the hypothesis of functional competence of sentinel lymph nodes based on the prevalence of the studied immune cells. The density of FOXP3+ lymphocytes showed association with progression and survival, but none of the immune markers emerged as independent prognostic factor. These results, together with our previous findings on the prognostic value of activated T cells and mature DCs infiltrating primary melanomas, suggest that immune cells residing in the primary tumor, rather than those in sentinel lymph nodes, may be crucial in the development of antitumor immune response reflected in influencing the outcome of the disease. This work was supported by the National Scientific Research Fund OTKA 72836. *The first two authors contributed equally to the work.

Citation Format: Anita Mohos, Timea Sebestyen, Gabriella Liszkay, Vanda Plotar, Szabolcs Horvath, Istvan Gaudi, Andrea Ladanyi. Immune cell profile of sentinel lymph nodes in melanoma patients . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 453. doi:10.1158/1538-7445.AM2013-453
Abstract 4538: A novel assay platform for the detection of kinase-inhibitor binding in intact mammalian cells.
Authors: Saharia Abhi , Raab William , Bassoni Daniel , Hunt Jeremy , Treiber Daniel K. , Wehrmann Tom
Published: August 2014

Kinase binding assays have become an integral part of inhibitor characterization. These tools have been applied in KINOME-wide screening systems as well as the detailed characterization of inhibitor residence time for lead optimization and MOA studies. However in cells, assays are limited to monitoring kinase activity and downstream substrate phosphorylation events. Although these types of cellular assays are critical to move programs forward, their ability to characterize inhibitor function is limited, requires targeted antibody reagents, and knowledge of specific substrates which are often not available. To circumvent these issues and apply the power of binding assays to a cellular format we have devised a method for monitoring compound binding to kinase targets in intact mammalian cells termed InCELL Hunter. The assay is shown to retain the benefits of cellular assays such as assessing compound permeability, toxicity, and target engagement; while exhibiting the generic applicability of a kinase binding assay. The InCELL hunter assay correctly describes type I, type II, and Type III inhibitors with the expected rank order cellular potencies. The assay is activity and substrate independent making it amenable to previously intractable targets and provides novel cellular information that complements both biochemical and existing cellular formats.

Citation Format: Abhi Saharia, William Raab, Daniel Bassoni, Jeremy Hunt, Daniel K. Treiber, Tom Wehrmann. A novel assay platform for the detection of kinase-inhibitor binding in intact mammalian cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4538. doi:10.1158/1538-7445.AM2013-4538
Abstract 4541: Highly sensitive kinase assays combining Carna Biosciences QSS Assist ELISA reagents with the Meso Scale Discovery MULTI-ARRAY platform.
Authors: Jacobi Carsten , Schilb Alain , Kawase Yusuke , Kirii Yasuyuki , Boldyreff Brigitte
Published: August 2014

We developed a sensitive kinase asay using a QSS Assist ELISA kinase assay kit from Carna Biosciences, Inc. and the detection method from Meso Scale Discovery. The QSS Assist ELISA kit is optimized for screening of compounds. Comparing to most other kinase assay kits, QSS Assist ELISA kits are available for both peptide and protein substrates and several ELISA kits developed by Carna Biosciences employ a naturally occurring protein substrate, e.g. MAP kinases for MAPK kinases.

Detection of kinase activity is achieved with a primary anti-phosphoserine/threonine or tyrosine antibody and a secondary anti-Ig-antibody. Commonly, such secondary antibodies are coupled to horseradish peroxidase (HRP), which allows for signal detection by adding a color reagent, i.e. an HRP substrate which can be detected upon reaction by measuring optical density. Here we applied an MSD SULFO-TAG labeled secondary antibody. These antibodies emit light upon electrochemical stimulation initiated at the electrode surfaces of MULTI-ARRAY microplates, and thus detect the primary antibody with ultimate sensitivity without direct labeling it.

We have sucessfully miniaturized the highly sensitive assay and this new assay technology has been validated with published kinase inhibitors from AstraZeneca and Tocris Bioscience.

Citation Format: Carsten Jacobi, Alain Schilb, Yusuke Kawase, Yasuyuki Kirii, Brigitte Boldyreff. Highly sensitive kinase assays combining Carna Biosciences QSS Assist ELISA reagents with the Meso Scale Discovery MULTI-ARRAY platform. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4541. doi:10.1158/1538-7445.AM2013-4541
Abstract 4551: Identification of TRPM7 kinase inhibitors.
Authors: Kaoud Tamer S. , Park Jihyun , Mitra Shreya , Tavares Clint D.J. , Tseng Anna , Dalby Kevin N.
Published: August 2014

TRPM7 (transient receptor potential melastatin 7) encodes a calcium permeable non-selective cation channel implicated in cell adhesion and magnesium homoeostasis. The TRPM7 gene encodes for a protein kinase whose activity is linked to the control of actomyosin contractility. TRPM7 mediates adhesion and migration of MDA-MB-231 breast cancer cells and recently has been reported to promote breast tumor cell metastasis. The lack of cell-permeable pharmacological inhibitors of the kinase domain represent a barrier to fully understanding kinase function. Herein, we describe the discovery of several compounds that target TRPM7 kinase activity and which were shown to decrease the binding of Myosin IIB to TRPM7 in HEK293 cells transfected with pCMV6-TRPM7. Magnesium starvation, which promotes TRPM7 kinase activity, induces phosphorylation of eEF2. Treatment of cells with several of the compounds decreased eEF2 phosphorylation under conditions of magnesium starvation, consistent with the notion that they inhibit TRPM7 kinase activity in cells.

Citation Format: Tamer S. Kaoud, Jihyun Park, Shreya Mitra, Clint D.J. Tavares, Anna Tseng, Kevin N. Dalby. Identification of TRPM7 kinase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4551. doi:10.1158/1538-7445.AM2013-4551
Abstract 4558: Discovery and mechanistic studies of novel class of CXCR2 inhibitors.
Authors: Ha Helen , Neamati Nouri
Published: August 2014

Background: Chemokine receptor, CXCR2 is expressed on various immune cells and is essential for neutrophil recruitment and angiogenesis at sites of acute and chronic inflammation caused by tissue injury or infection. Due to its role in inflammation, it has been implicated in a number of inflammation-mediated diseases such as psoriasis, arthritis, COPD, cystic fibrosis, and asthma. More recently, it is also implicated in various types of cancer. CXCR2 and its ligands are up-regulated in cancer cells as well as the tumor microenvironment and have been shown to promote tumor growth, angiogenesis and invasiveness. Though pharmaceutical companies have pursued the development of CXCR2-specific small-molecule inhibitors as anti-inflammatory agents within the two past decades, there are currently no clinically approved CXCR2 inhibitors.

Methodology/Principal Findings: Using a high-throughput, cell-based, CXCR2 assay, we screened an in-house library of structurally diverse compounds and identified a novel class of CXCR2 biased ligand. Our lead compound, CX797, inhibited IL8-mediated cyclic AMP signaling and receptor degradation, while specifically up-regulating IL8-mediated β-arrestin recruitment and MAP kinase signaling. CX797 also inhibited Jurkat cell proliferation via G0/G1 cell cycle arrest.

Conclusions/Significance: In our studies we have identified a novel class of CXCR2 ligand, CX797, and the first to report biased CXCR2 ligand signaling. Mechanistic studies of CX797 and previously reported CXCR2 inhibitor, SB265610, show these two classes have distinct mechanism of action on CXCR2. CX797 is a potential candidate for further optimization and development for various inflammatory diseases and CXCR2-expressing cancer.

Citation Format: Helen Ha, Nouri Neamati. Discovery and mechanistic studies of novel class of CXCR2 inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4558. doi:10.1158/1538-7445.AM2013-4558
Abstract 4591: Whole genome sequencing of SHH medulloblastomas predicts molecular groups of responders and non-responders to SMO-inhibition .
Authors: Kool Marcel , Jones David TW , Jaeger Natalie , Northcott Paul A. , Hovestadt Volker , Schueller Ulrich , Remke Marc , Cho Yoon-Jae , Pomeroy Scott , Taylor Michael D. , Eils Roland , Korshunov Andrey , Lichter Peter , Pfister Stefan M.
Published: August 2014

Recently, we and others analyzed large series of pediatric medulloblastomas (MB) and identified a number of recurrent aberrations in each of the four major subgroups (WNT, SHH, Group 3 and Group 4). However, due to their infrequent occurrence not much is known about genetic aberrations in adult MBs. We now have analyzed a large series of adult SHH MBs (age ≥ 16) by whole genome sequencing and compared them with pediatric SHH MBs. We sequenced tumor and blood of 26 adult SHH MBs and compared them with 30 pediatric SHH MBs. A replication cohort of 28 adult and 29 pediatric SHH MBs was sequenced for 414 genes by targeted sequencing. We also performed expression profiling and DNA methylation analyses on most of these cases. Whole genome sequencing revealed a clear correlation between mutation rate and age of the patient (r = 0.69). Furthermore, the genomic data showed that SHH MBs can be split up in three distinct subgroups: infants, children and adults. They all have mutations in the SHH pathway, mostly in PTCH1 (infants and adults), SUFU (infants) and SMO (adults). Children almost completely lack the classical upstream SHH pathway mutations but instead have more downstream aberrations such as MYCN and GLI2 mplifications, and have frequent TP53 mutations, often in the germline, whilst all of these were extraordinarily rare in infants and adults. We also identified several recurrent mutations in adult MBs that were not found in pediatric MBs. Moreover, the three SHH subgroups were different in their transcriptome and methylome with the TP53 mutated SHH MBs in children being more similar to adult MBs. Collectively, this data will help to better understand the biology of pediatric and adult SHH-medulloblastoma, but most importantly may help selecting patients for targeted SHH inhibition and new treatment strategies for TP53 mutated SHH MBs in a clinical setting.

Citation Format: Marcel Kool, David TW Jones, Natalie Jaeger, Paul A. Northcott, Volker Hovestadt, Ulrich Schueller, Marc Remke, Yoon-Jae Cho, Scott Pomeroy, Michael D. Taylor, Roland Eils, Andrey Korshunov, Peter Lichter, Stefan M. Pfister. Whole genome sequencing of SHH medulloblastomas predicts molecular groups of responders and non-responders to SMO-inhibition . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4591. doi:10.1158/1538-7445.AM2013-4591