bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2437: Crystal structures of CARM1 bound to sinefungin and diverse peptide substrates
Authors: Boriack-Sjodin Ann , Jin Lei , Jacques Suzanne L. , Drew Allison , Porter Scott Margaret , Ribich Scott , Moradei Oscar
Published: August 2015

Co-activator-associated arginine methyltransferase 1 (CARM1) is a protein arginine N-methyltransferase (PRMT) enzyme that has been implicated in a variety of cancers including AML (1) and breast (2), prostate (3), lung (4) and colorectal (5) carcinomas. CARM1 is known to methylate H3 histones and non-histone substrates including p300/CBP, AIB1/SRC-3 and PABP1 (6). To date, several crystal structures of CARM1 have been solved, including structures with small molecule inhibitors (7), but no ternary structures with nucleotide and peptide substrate have been reported. Here, the crystal structures of human CARM1 with the SAM mimic sinefungin (SFG) and three different peptide sequences from histone H3 and PAPB1 are presented, and both non-methylated and singly-methylated arginine residues are exemplified. Extensive interactions are seen with residues Glu266, Glu257, and His414 and the substrate arginine side chain. Two key hydrogen bonds are made by the side chain of Asn161 with the backbone carbonyl of the P’1 peptide residue and the backbone NH of the P’3 peptide residue. The carbonyl of the P1 peptide residue engages the protein through water mediated hydrogen bonds. These structures show how the CARM1 binding site is capable of accommodating a variety of peptide sequences. Comparisons to known CARM1 complexes with small molecules provide additional insights into inhibitor design.

1. Vu, L. P. et al, PRMT4 blocks myeloid differentiation by assembling a methyl-RUNX1-dependent repressor complex. Cell Rep 2013, 5 (6), 1625-38.

2. Al-Dhaheri, M. et al, CARM1 is an important determinant of ERalpha-dependent breast cancer cell differentiation and proliferation in breast cancer cells. Cancer Res 2011, 71 (6), 2118-28.

3. Kim, Y. R. et al, Differential CARM1 expression in prostate and colorectal cancers. BMC Cancer 2010, 10, 197.

4. Elakoum, R. et al, CARM1 and PRMT1 are dysregulated in lung cancer without hierarchical features. Biochimie 2014, 97, 210-8.

5. Ou, C. Y. et al, A coactivator role of CARM1 in the dysregulation of beta-catenin activity in colorectal cancer cell growth and gene expression. Mol Cancer Res 2011, 9 (5), 660-70.

6. Bedford M.T.; Clarke S.G. Protein arginine methylation in mammals: who, what, and why. Mol Cell 2009, 33, 1-13.

7. Sack, J.S et al, Structural basis for CARM1 inhibition by indole and pyrazole inhibitors. Biochem J 2011, 436, 331-9.

Citation Format: Ann Boriack-Sjodin, Lei Jin, Suzanne L. Jacques, Allison Drew, Margaret Porter Scott, Scott Ribich, Oscar Moradei. Crystal structures of CARM1 bound to sinefungin and diverse peptide substrates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2437. doi:10.1158/1538-7445.AM2015-2437
Abstract 2443: Optides (optimized peptides), a new knottin-based cancer drug discovery platform
Authors: Olson James M. , Strong Roland , Mehlin Christopher , Correnti Colin
Published: August 2015

Some of the highest value targets in cancer therapeutics involve protein:protein interactions that are difficult to inhibit with small molecule therapeutics. Peptide drug candidates offer an alternative, but many peptide drugs have poor pharmacokinetic properties and some are immunogenic. Knottin peptides have long been promoted as scaffolds for human drug discovery efforts because the exquisitely tight knot formed by disulfide bridges creates resistance to proteolytic and other forms of degradation. Furthermore, some knottins cross the blood brain barrier, the gut wall, or cell membranes which is ideal for certain targets. Unfortunately, creating knottins synthetically or in bacteria results in a large number of disulfide isomers, which has hampered research. We have created a mammalian expression system that enables production of most knottin scaffolds and variants of native knottins encoded in plant and animal genomes. The platform routinely produces approximately 10 mg/liter of near pure naked peptide, which is sufficient to conduct in vivo studies without major scale-up. The system is endotoxin free. In addition to building the platform, we identified over 200,000 new potential knottin peptides in various genomes, creating a rich collection of scaffolds from which to establish diversity libraries of native and optimized variants. These candidates have potential for targeting therapeutics to cancer cells as a solid tumor penetrating alternative to antibody drug conjugates. Optides also have potential to be developed as anti-cancer drugs themselves. The fact that knottin peptides are not substrates for resistance mechanisms such as p-glycoprotein pumps underscores the potential importance of this class of drugs for those with previously treated cancer.

Citation Format: James M. Olson, Roland Strong, Christopher Mehlin, Colin Correnti. Optides (optimized peptides), a new knottin-based cancer drug discovery platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2443. doi:10.1158/1538-7445.AM2015-2443
Abstract 2447: Development of Hsp90 paralog specific inhibitors with mitochondrial drug delivery system
Authors: Kang Byoung Heon
Published: August 2015

The mitochondrial pool of Hsp90 and its mitochondrial paralog, TRAP1, suppresses cell death and reprograms energy metabolism in cancer cells; therefore, Hsp90 and TRAP1 have been suggested as target proteins for anticancer drug development. Here, we report that the actual target protein in cancer cell mitochondria is TRAP1, not Hsp90, and current Hsp90 inhibitors cannot effectively inactivate TRAP1 due to insufficient accumulation in the mitochondria. To develop mitochondrial TRAP1 inhibitors, we determined the crystal structures of human TRAP1 complexed with Hsp90 inhibitors. The isopropyl amine of the Hsp90 inhibitor PU-H71 was replaced with the mitochondria-targeting moiety triphenylphosphonium to produce SMTIN-P01. SMTIN-P01 showed a different mode of action from the non-targeted PU-H71, as well as much improved cytotoxicity to cancer cells. In addition, we determined the structure of a TRAP1-adenylyl-imidodiphosphate (AMP-PNP) complex. Based on comparative analysis of TRAP1 structures, we propose a molecular mechanism of ATP hydrolysis that is crucial for chaperone function.

Citation Format: Byoung Heon Kang. Development of Hsp90 paralog specific inhibitors with mitochondrial drug delivery system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2447. doi:10.1158/1538-7445.AM2015-2447
Abstract 2451: The nuclear-targeted gold nanoparticles inhibit the tumor growth in mouse model as potential anticancer agents
Authors: Peng Xianghong , Macke Megan A. , Shin Hyung Ju C , Nannapaneni Sreenivas , Chen Nelson , Kim Sungjin , Chen Zhuo (Georgia) , El-Sayed Mostafa A , Shin Dong
Published: August 2015

The use of gold nanoparticles (AuNPs) as chemotherapeutic agents, based on their intrinsic antineoplastic properties is becoming an increasingly exciting possibility for the treatment of cancer. The specific targeting of AuNPs to the nucleus of malignant cells can be achieved via peptide functionalization of the nanoparticle surface, resulting in DNA damage and subsequent apoptosis. Here, we utilized these novel AuNPs for systemic delivery in vivo and determined their efficacy as chemotherapeutic agents. In addition, we assessed their effects on overall health in order to characterize their systemic toxicity. AuNPs (30 nm in diameter) were functionalized with an Arg-Gly-Asp (RGD) peptide sequence, providing tumor cell specific targeting, as well as a Lys-Lys-Lys-Arg-Lys nuclear localization sequence (NLS) peptide. These novel nuclear-targeting AuNPs were delivered via tail vein injection in nude mice bearing Tu212 tumors. Their in vivo therapeutic efficacy was evaluated, along with assessing their systemic toxicity. The AuNPs, specifically targeted to the tumor cell nucleus, exhibit circulation half-life of 4.1 h, which is exceptionally greater than common chemotherapeutic agents and, although they exhibit uptake by organs of the reticuloendothelial system (RES) (e.g. liver and spleen), they also accumulate in the tumor. In addition, when systemically delivered at low doses (5 nM AuNPs) these novel targeted AuNPs show significant tumor regression (via DNA damage and subsequent apoptosis) when compared to their non-targeting counterparts (P = 0.004), with no observed toxicity in terms of hepatic function, renal function, or body weight. In conclusion, by delivering AuNPs to the nucleus of tumor cells, at low doses in vivo, high therapeutic efficacy is achieved with minimal side effects, indicating the potential for the use of these novel nuclear-targeted AuNPs as chemotherapeutic agents.

Citation Format: Xianghong Peng, Megan A. Macke, Hyung Ju C Shin, Sreenivas Nannapaneni, Nelson Chen, Sungjin Kim, Zhuo (Georgia) Chen, Mostafa A El-Sayed, Dong Shin. The nuclear-targeted gold nanoparticles inhibit the tumor growth in mouse model as potential anticancer agents. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2451. doi:10.1158/1538-7445.AM2015-2451
Abstract 246: Phase 1 study: Ipilimumab (anti CTLA-4) in combination with Lenalidomide in patients with advanced cancers
Authors: Sakamuri Divya , Betancourt Cuellar Sonia L. , Glitza Isabella C. , Fu Siqing , Wheler Jennifer J. , Falchook Gerald S. , Tsimberidou Apostolia M. , Hong David S. , Subbiah Vivek , Fanale Michelle A. , Cabanillas Maria E. , Meric-Bernstam Funda , Janku Filip
Published: August 2015

Background: Ipilimumab, a cytotoxic T-lymphocyte antigen 4 (CTLA-4) blocking antibody potentiates an antitumor T-cell response. Lenalidomide, a derivative of thalidomide possesses immunomodulatory, antiangiogenic, and antineoplastic properties and thereby inhibits the growth of blood vessels and exhibits an anti-inflammatory effect. The potential synergistic effect of stimulating one's own immune system to attack cancer in combination with depriving further tumor growth provides rationale for combining Ipilimumab and Lenalidomide.

Methods: We designed a phase I study (3+3 design) to determine the safety of Ipilimumab (1.5-3mg/kg IV q 28 days x 4) and Lenalidomide (10-25mg PO daily x 21 q 28 days) until disease progression or unacceptable toxicity in advanced cancers. Endpoints included maximum tolerated (MTD), dose limiting toxicities (DLT), safety, and response per immune response criteria (iRC).

Results: To date, 21 patients (men, n = 8; women, n = 13), median age 56 (19-75) years, median of 3 (0-6) prior therapies (including 3 patients with prior ipilimumab) were enrolled in 3 of 5 dose levels ranging from 1.5-3mg of Ipilimumab and Lenalidomide 10-15 mg. The MTD has not been reached yet. DLT included grade (G) 3 macular rash in 2 patients (dose levels 2 and 3) and G3 pancreatitis (dose level 2). Other G3/4 drug related toxicities included G4 hypopituitarism (n = 1) and G3 pulmonary embolism (n = 1). Of 21 treated patients, 11 (52%) patients had stable disease. The median change in size of target lesions per iRC was 13% (-44% to +86%) with 5 (24%) of patients with at least -10% shrinkage (Hodgkin lymphoma, n = 2; melanoma, n = 1; follicular thyroid carcinoma, n = 1; teratoma, n = 1) lasting for 0.9 to 12.3+ months. The median time-to-treatment failure was 2.1 months (95% CI 1.4-2.8).

Conclusion: Ipilimumab and Lenalidomide have manageable side effect profile and demonstrate possible activity in patients with refractory Hodgkin lymphoma, melanoma, follicular thyroid cancer and teratoma.

Citation Format: Divya Sakamuri, Sonia L. Betancourt Cuellar, Isabella C. Glitza, Siqing Fu, Jennifer J. Wheler, Gerald S. Falchook, Apostolia M. Tsimberidou, David S. Hong, Vivek Subbiah, Michelle A. Fanale, Maria E. Cabanillas, Funda Meric-Bernstam, Filip Janku. Phase 1 study: Ipilimumab (anti CTLA-4) in combination with Lenalidomide in patients with advanced cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 246. doi:10.1158/1538-7445.AM2015-246
Abstract 2486: Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar
Authors: Zhu Zhongyu , Boopathy Ramakrishnan , Li Jinyu , Prabakaran Ponraj , Colantonio Simona , Feng Yang , Wang Yanping , Dyba Marzena A. , Dimitrov Dimiter S.
Published: August 2015

Conjugation of small molecule drugs to specific sites on the antibody molecule has been increasingly used for the generation of relatively homogenous preparations of antibody-drug conjugates (ADCs) with physicochemical properties similar or identical to those of the naked antibody. We previously developed a method for conjugation of small molecules to glycoproteins through existing glycans by using an engineered glycotransferase and a chemically reactive sugar as a handle to conjugate a small molecule. Here, for the first time, we report the use of this method with some modifications to generate an ADC from a monoclonal antibody, m860, which we identified from a human naïve phage display Fab library by panning against the extracellular domain of human HER2. M860 bound to cell surface-associated HER2 with affinity comparable to that of trastuzumab (Herceptin®), but to a different epitope. The ADC m860 was generated by enzymatically adding a reactive keto-galactose to galactose-free m860 using an engineered glycotransferase and conjugating the reactive m860 to aminooxy auristatin F with a non-cleavable linker. It exhibited exceptional serum stability and potent and specific cell-killing activity against HER2 positive cancer cells, including trastuzumab-resistant breast cancer cells. This unique ADC may have utility as a potential therapeutic for HER2 positive cancers alone or in combination with other drugs. Our results also validate the keto-galactose/engineered glycotransferase method for generation of functional ADCs, which could potentially also be used for preparation of ADCs targeting other disease markers.

This project was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, and by federal funds from the National Cancer Institute, NIH, under contract N01-CO-12400, HHSN261200800001E as well as by the SU2C-St.Baldrick's Foundation.

Citation Format: Zhongyu Zhu, Ramakrishnan Boopathy, Jinyu Li, Ponraj Prabakaran, Simona Colantonio, Yang Feng, Yanping Wang, Marzena A. Dyba, Dimiter S. Dimitrov. Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2486. doi:10.1158/1538-7445.AM2015-2486
Abstract 2495: STING agonists formulated into cancer vaccines (STINGVAX) can cure established tumor resistant to immune checkpoint blockade by activating NK cells
Authors: Kim Young , Pardoll Drew , Fu Juan , Dubensky Tom
Published: August 2015

Stimulator of Interferon Genes (STING) is a cytosolic receptor that senses both exogenous and endogenous cytosolic cyclic dinucleotides, activating TBK-1/IRF-3, NF-κB, and STAT6 signaling pathways to induce robust type I interferons and proinflammatory cytokines. Cyclic dinucleotide ligands were formulated with GM-CSF-producing cellular cancer vaccines - termed STINGVAX - that demonstrated potent in vivo anti-tumor efficacy in multiple therapeutic models of established cancer. We found that rationally designed synthetic CDN derivative molecules, including one with an Rp,Rp dithio diastereomer and non-canonical c[A(2′,5′)pA(3′,5′)p] phosphate bridge structure conferred significantly enhanced therapeutic anti-tumor efficacy in multiple aggressive therapeutic models of established cancer in mice. Anti-tumor activity was STING-dependent and correlated with increased activation of dendritic cells and tumor antigen-specific CD8+ T cells. Tumors from STINGVAX treated mice demonstrated dramatic PD-L1 upregulation, which was associated with tumor infiltrating CD8+IFNγ+ T-cells. When combined with PD-1 blockade, STINGVAX induced regression of palpable, poorly immunogenic tumors that did not respond to PD-1 blockade alone. We found that the STINGVAX's anti-tumor efficacy is NK cell dependent, and demonstrated that CDN are potent activators of IFNg in NK cells.

Citation Format: Young Kim, Drew Pardoll, Juan Fu, Tom Dubensky. STING agonists formulated into cancer vaccines (STINGVAX) can cure established tumor resistant to immune checkpoint blockade by activating NK cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2495. doi:10.1158/1538-7445.AM2015-2495
Abstract 2514: Activity of a novel Foxp3-tumor cell vaccine in a murine model of renal cell carcinoma
Authors: Shen Li , Orillion Ashley , Adelaiye Remi , Ciamporcero Eric , Ramakrishnan Swathi , Pili Roberto
Published: August 2015

Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults and represents 80% of the cases. Treatment options for patients with advanced disease are limited. Targeted molecular therapies have shown survival benefit but most patients develop therapy resistance overtime. Preclinical and clinical studies indicate that immunotherapies are effective and may play a role in the treatment of advanced RCC. However, the response rate is ∼ 20%, with a limited number of patients achieving durable remission. A major barrier to vaccine therapy is the presence of immunosuppressive cell populations including regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). In the current study, we designed a tumor cell and Tregs dual-targeting vaccine by inducing Foxp3 expression in tumor cells, and tested this vaccine along with an unmodified vaccine in an orthotopic murine model of RCC, RENCA. We observed that both RENCA cell vaccine and RENCA Foxp3 cell vaccine prevented tumor growth in 6 out 10 animals. Interestingly, in an intervention setting, the RENCA Foxp3 tumor cell vaccine was superior to the RENCA tumor cell vaccine with a significant anti-tumor activity (45% inhibition of tumor weights) (p<0.01). Moreover, RENCA Foxp3 tumor cell vaccine reduced the number of tumor infiltrating Tregs (42% reduction, p<0.05) and Treg Foxp3 expression both in peripheral blood and tumor infiltrates. In summary, our results suggest that a strategy targeting Tregs along with vaccine therapy or a dual targeting vaccine strategy may be beneficial in established RCC. We also observed that suppressive myeloid cell (SMCs) populations dominate the established tumor microenvironment and we are currently testing the combination of the dual-targeting vaccine with a SMC-targeting agent.

Citation Format: Li Shen, Ashley Orillion, Remi Adelaiye, Eric Ciamporcero, Swathi Ramakrishnan, Roberto Pili. Activity of a novel Foxp3-tumor cell vaccine in a murine model of renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2514. doi:10.1158/1538-7445.AM2015-2514
Abstract 2546: HDAC and Hsp90 inhibition as therapeutic strategy for translocation renal cell carcinoma
Authors: Ku Sheng-Yu , Ramakrishnan Swathi , Ciamporcero Eric , Xu Bo , Azabdaftari Gissou , Cheney Richard , Pili Roberto
Published: August 2015

Background: Xp11.2 translocation renal cell carcinoma (tRCC) is an aggressive subtype of kidney cancer, which mainly occurs in young patients. The prevalence of this disease is about 30% in pediatric RCC, but ∼1% in adult RCC cases. Patients with tRCC are refractory to standard therapies for clear cell RCC. Therefore, there is no efficient treatment for this type of kidney cancer. The tRCC harbors gene fusions involving the TFE3 transcription factor on chromosome X with several partner genes, leading to TFE3 elevation in the cell nucleus, but its function remains unclear. We have successfully established a cell line and patient-derived xenograft from a tRCC patient to develop rational therapeutic strategies and understand the tumor biology.

Methods: We have utilized a feeder layer containing a ROCK inhibitor to favorably generate a cell line, 277T, from a young tRCC patient. Also, we have implanted tumor samples from the patient subcutaneously into SCID mice to establish a patient-derived xenograft model, RP-R-07, and continuously maintain tumor growth in vivo. Cells were treated with different drugs, and then assessed by MTT assay and trypan blue staining for growth inhibition and cell death investigations. TFE3 expression in cells and tumors were detected by immunofluorescence and immunohistochemistry. The combined drug effects were determined by CalcuSyn analysis.

Results: Our preliminary results demonstrated that TFE3 is predominately expressing in the nucleus of 277T cells and RP-R-07 tumor model compared to other subtypes of RCC by immunofluorescence and immunohistochemistry, respectively. Further, we treated 277T cells with several cytotoxic drugs showing that the cells didn't respond to docetaxel, doxorubicin, rapamycin, or a PI3K-mTOR inhibitor, but were sensitive to the Hsp90 inhibitor AUY922, and the HDAC inhibitor LBH589. Moreover, combining both AUY922 and LBH589 induced greater cell death compared to each single treatment (73% vs 41% for LBH589; 73% vs 19% for AUY922). In addition, an in vivo experiment indicated that LBH589 did inhibit tumor growth.

Conclusions: Overall our results indicate that tRCC is sensitive to HSP90 and HDAC inhibitors both in vitro and in vivo. The combination of LBH589 and AUY922 represents a promising strategy for treating tRCC

Citation Format: Sheng-Yu Ku, Swathi Ramakrishnan, Eric Ciamporcero, Bo Xu, Gissou Azabdaftari, Richard Cheney, Roberto Pili. HDAC and Hsp90 inhibition as therapeutic strategy for translocation renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2546. doi:10.1158/1538-7445.AM2015-2546
Abstract 2566: Combination of taxanes with mesothelin-targeted immunotoxin RG7787 induces synergistic killing of pancreatic cancer
Authors: Alewine Christine Campo , Kolyvas Emily , Boslett Klaus , Pastan Ira
Published: August 2015

Pancreatic cancer has a poor prognosis with a 5 year median overall survival of just 6%. Most pancreatic adenocarcinomas express the cells surface antigen mesothelin (MSLN). RG7787 is a cytolytic fusion protein that uses an anti-mesothelin (MSLN) antibody Fab to target a modified, deimmunized Pseudomonas exotoxin A cytotoxic payload to cells that express MSLN. The immunotoxin is internalized by cells, releasing the toxin to the cytosol, where it enzymatically modifies elongation factor-2 causing a global halt in protein synthesis. We have previously observed that combination of paclitaxel with RG7787 significantly enhances anti-tumor effect in a KLM1 xenograft model of pancreatic cancer. Here, we report that co-treatment with nab-paclitaxel induces durable complete regressions in this model as well as explore the mechanisms underlying the favorable interaction of RG7787 with taxanes. We demonstrate that the enhanced killing with the combination can also be observed in vitro. Co-treatment lowers the dose of RG7787 required to achieve complete cell killing to the physiologic range. This favorable interaction requires the toxin portion of the molecule and cannot be replicated by treating with the anti-MSLN Fab alone. The timing of treatment with the two drugs is not critical, as similar cytotoxic effects are observed when taxane is given 24 hrs before, 24 hrs after or simultaneously with RG7787. Addition of taxane does not increase immunotoxin-mediated protein synthesis inhibition, nor can combination of taxane with the protein synthesis inhibitor cycloheximide reproduce the enhanced killing seen with the taxane/ RG7787 combination. Further studies exploring the critical factors in this interaction are ongoing.

Citation Format: Christine Campo Alewine, Emily Kolyvas, Klaus Boslett, Ira Pastan. Combination of taxanes with mesothelin-targeted immunotoxin RG7787 induces synergistic killing of pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2566. doi:10.1158/1538-7445.AM2015-2566
Abstract 26: Suppression of E-cadherin mediates gallotannin-induced apoptosis in Hep G2 hepatocelluar carcinoma cells
Authors: Kwon Hee Young , Jung Ji Hoon , Lee Hyun Joo , Jeong Myoung Seok , Jung Deok-Beom , Kim Bonglee , Lee Hyemin , Kim Sung-Hoon
Published: August 2015

Though gallotannin was known to have anti-oxidant and antitumor activity, the underlying antitumor mechanism of gallotannin still remains unclear. Thus, in the present study, antitumor mechanism of gallotannin was elucidated in hepatocellular carcinoma cells. Gallotannin significantly exerted cytotoxicity against Hep G2 and Chang hepatocellular carcinoma cells with the accumulation of the sub-G1 population and increase of terminal deoxynucleotidyltransferasedUTP nick end labeling (TUNEL) positive cells as an apoptotic feature. Also, gallotannin attenuated the expression of pro-caspase9, pro-caspase3, Bcl2 and integrin β1 and cleaved poly(ADP)-ribose polymerase (PARP) in Hep G2 and Chang cancer cells. Furthermore, gallotannin suppressed cell repair motility by wound healing assay and also inhibited cell adhesion in Hep G2 cells. Of note, gallotannin attenuated the expression of epithelial cadherin (E-cadherin) to form cell-cell adhesion from the early stage, and also beta-catenin at late phase in Hep G2 cells. Consistently, Immunofluorescence assay showed that E-cadherin or β-catenin expression was suppressed in a time dependent manner by gallotannin. Furthermore, silencing of E-cadherin by siRNA transfection method enhanced PAPR cleavage, caspase 3 activation and sub G1 population and attenuated the cell adhesion induced by gallotannin in Hep G2 cells. Overall, our findings demonstrate that the disruption of cell adhesion junction by suppression of E-cadherin mediates gallotannin enhanced apoptosis in Hep G2 liver cancer cells.

Citation Format: Hee Young Kwon, Ji Hoon Jung, Hyun Joo Lee, Myoung Seok Jeong, Deok-Beom Jung, Bonglee Kim, Hyemin Lee, Sung-Hoon Kim. Suppression of E-cadherin mediates gallotannin-induced apoptosis in Hep G2 hepatocelluar carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 26. doi:10.1158/1538-7445.AM2015-26
Abstract 2609: Disruption of CAS scaffolds: A novel mechanism for microtubule targeting agents
Authors: Dybdal-Hargreaves Nicholas F. , Rohena Cristina C. , Risinger April L. , Mooberry Susan L.
Published: August 2015

Microtubule targeting agents (MTAs) are an important class of anticancer drugs. While their anticancer mechanisms were considered exclusively as antimitotic for decades, evidence now suggests that the ability of MTAs to inhibit functions of interphase microtubules likely plays an essential role in their clinical anticancer actions. Interphase microtubules are responsible for intracellular trafficking of many constituents including proteins involved in signal transduction. Efficient signal transduction requires precise coordination of signaling partners through their appropriate localization. Scaffold proteins regulate signal transduction pathways by assembling signaling components into complexes both temporally and spatially. Assembly of scaffold complexes can be dependent on a functional microtubule network. Several scaffold proteins have been identified with importance in breast cancer, including BCAR1, a member of the crk associated substrate (CAS) family of scaffolds. BCAR1 expression in breast cancer is associated with resistance to anti-estrogens. We hypothesized that the ability of diverse MTAs to interrupt the function of these signaling scaffolds could contribute to their activities in breast cancer cells. The effects of eribulin, vinorelbine, paclitaxel, docetaxel, and ixabepilone on the localization and associated signaling of BCAR1 was evaluated. The MTAs disrupted the localization of BCAR1 and differential effects were noted among the MTAs. There were also distinct effects of the agents on BCAR1 associated signaling. To determine whether BCAR1 expression was related to sensitivity of breast cancer cell lines to specific MTAs we separated the breast cancer cell lines based on their ability to initiate cytotoxic or only antiproliferative effects and evaluated them for BCAR1 expression. Two cell lines with the highest BCAR1 expression were resistant to the cytotoxic effects of each MTA of this panel. Thus, high levels of BCAR1 in breast cancer cells can overcome the ability of diverse MTAs to disrupt scaffold function and lead to resistance. Our results suggest that the ability of MTAs to interrupt BCAR1 scaffolds represents a new mechanism of action for these drugs. However, MTA disruption of the BCAR1 CAS scaffold complexes can be overcome by high BCAR1 expression. Funding for the work was provided by Eisai Inc.

Citation Format: Nicholas F. Dybdal-Hargreaves, Cristina C. Rohena, April L. Risinger, Susan L. Mooberry. Disruption of CAS scaffolds: A novel mechanism for microtubule targeting agents. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2609. doi:10.1158/1538-7445.AM2015-2609
Abstract 2614: Targeting heat shock protein 90 with Ganetespib for the treatment of lymphoma
Authors: Tu Zhigang , Liu Hanqing , Zhao Jingjing , Zhang Peishan , Liang Zhiquan , Ruan Lingling , Lu Yongjin , Shang Dongsheng , Sun Ying , Xu Lili , Chen Keping , Danzeng Quji
Published: August 2015

Effective treatments for lymphoma remain a serious unmet medical need: the incidence of lymphoma continues to rise, and lymphoma tumors can relapse in patients who initially respond to treatment. Heat shock protein 90 (HSP90) has become an attractive therapeutic target in treating many many cancers, including blood cancers. In our study, we show that ganetespib, a clinically promising and actively investigated second-generation HSP90 inhibitor, dramatically reduced growth of a couple of different kinds of lymphoma, such as mantle cell lymphoma and Burkitt's lymphoma in a dose-dependent manner. It induced G2/M cell-cycle arrest and apoptosis in all studied cell lines, such as Ramos, Jeko-1, Granta-519, Raji, etc. Ganetespib significantly inhibited growth of xenograft tumors in vivo as a single agent or in combination with doxorubicin. Results of H&E staining, TUNEL assays, and immunohistochemistry staining of Ki-67 revealed significant differences in ganetespib-treated tumors. Mechanistically, ganetespib caused pronounced decrease of expression of classic HSP90 client proteins. In summary, our data suggest that ganetespib can be potentially used for the molecularly targeted therapy of lymphoma patients.

Citation Format: Zhigang Tu, Hanqing Liu, Jingjing Zhao, Peishan Zhang, Zhiquan Liang, Lingling Ruan, Yongjin Lu, Dongsheng Shang, Ying Sun, Lili Xu, Keping Chen, Quji Danzeng. Targeting heat shock protein 90 with Ganetespib for the treatment of lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2614. doi:10.1158/1538-7445.AM2015-2614
Abstract 2641: Inhibition of Bcr-Abl in human leukemic cells with a coiled coil protein delivered by a leukemia-specific cell penetrating peptide
Authors: Bruno Benjamin J. , Lim Carol S.
Published: August 2015

The oncoprotein Bcr-Abl is the cause of chronic myeloid leukemia (CML). Current CML therapies target the tyrosine kinase domain of Bcr-Abl, but due to their non-curative nature, resistance to these drugs is common. Therefore, new treatments are needed for those patients refractive to available therapies. Bcr-Abl homo-oligomerization via its N-terminal coiled coil (CC) domain is required for tyrosine kinase activity. Our previous work has shown that it is possible to inhibit Bcr-Abl activity by targeting the CC domain with a altered version of the CC domain, known as CCmut3, delivered as a plasmid. In this study, CCmut3 is delivered to cells as a protein by utilizing a leukemia-specific cell-penetrating peptide (CPP). Here, recombinant CPP-CCmut3 was expressed, purified, and tested for its anti-oncogenic activity. CPP-CCmut3 was able to enter two leukemic cell lines (K562 and Ba/F3-P210) and inhibit Bcr-Abl activity as shown by induction of necrosis/apoptosis via 7-AAD/Annexin V staining, reduction of oncogenic potential in colony forming assays, reduction of cell proliferation, and inhibition of Bcr-Abl phosphorylation (kinase activity). Further, CPP-CCmut3 did not enter non-leukemic cell lines (HEK293 and MCF-7). While CPP-CCmut3 was able to enter the parental, non-leukemic Bcr-Abl- Ba/F3 pro-B cell line, it revealed no signs of activity in the assays performed, as expected. These results indicate the feasibility of using CPP-CCmut3 as a therapeutic against CML.

Citation Format: Benjamin J. Bruno, Carol S. Lim. Inhibition of Bcr-Abl in human leukemic cells with a coiled coil protein delivered by a leukemia-specific cell penetrating peptide. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2641. doi:10.1158/1538-7445.AM2015-2641
Abstract 2647: Anti-tumor activity of sHA8k, a HYAL1 hyaluronidase inhibitor, in bladder cancer cells
Authors: Jordan Andre R. , Chipollini Juan , Lopez Luis , Yates Travis , Lokeshwar Vinata
Published: August 2015

INTRODUCTION AND OBJECTIVE: Tumor cell-derived hyaluronidase HYAL1, which degrades hyaluronic acid (HA) into angiogenic fragments, promotes tumor growth and metastasis. HYAL-1 is an independent prognostic marker for predicting metastasis. Small molecular mass sulfated hyaluronic acid (sHA) derivatives (sHA8k) inhibit HYAL1 activity through a mixed inhibition mechanism. Antitumor activity of sHA8k and mechanism of action was evaluated in BCa models..

METHODS: Effect of sHA8k (0-40 ug/ml) on cell proliferation and apoptosis was examined in BCa cells (253J-Lung, HT1376, UMUC-3, T24, RT4) by cell counting and Cell Death ELISA kit. Matrigel invasion and Boyden chamber assays were used to test the effect of sHA8k on invasive activity. Effect of sHA on signaling, apoptosis cascade, HA receptor (CD44, RHAMM), EMT markers (β-catenin, E-cadherin, Snail, Twist) levels, was evaluated by Q-pCR and immunoblotting assays. Angiogenic fragment addition and mAkt transfection were performed to elucidate mechanism of action. Athymic mice bearing 253J-Lung xenografts were treated with sHA (25 and 50 mg/kg) by i.p. injection.

RESULTS: sHA8k inhibited proliferation, motility and invasion in BCa cells that expressed HYAL-1. At IC50 for HAase activity inhibition (∼ 20-μg/ml), sHA induced > 3-fold apoptosis and inhibited invasive activity of BCa cells. sHA8k induced caspase-3, -8, -9 activation, up-regulation of Fas, Fas-L, FADD, DR4, DR5 and E-cadherin. sHA8k downregulated CD44, RHAMM, bcl-2, phospho(p)-Akt, pGSK3β, pβcatenin(ser552), snail and Twist expression. Effect of sHA8k were attenuated by angiogenic HA fragments or overexpression of m-Akt and downregulation of CD44 and RHAMM mimicked sHA8k effects. sHA significantly inhibited 253J-Lung xenograft growth. The majority of the animals did not form palpable tumors at 50-mg/kg dose. No weight loss or serum and organ toxicity was observed in sHA treated animals. Biochemical analysis of tumors also showed the same alterations in Akt and EMT pathways as observed cell culture.

CONCLUSION: This is the first study that shows sHA8k, a small molecular mass HYAL-1 inhibitor has potent antitumor activity.

Support: R01 CA 72821-14 (VBL)

Citation Format: Andre R. Jordan, Juan Chipollini, Luis Lopez, Travis Yates, Vinata Lokeshwar. Anti-tumor activity of sHA8k, a HYAL1 hyaluronidase inhibitor, in bladder cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2647. doi:10.1158/1538-7445.AM2015-2647
Abstract 2649: Inhibition of mTOR, but not PI3K, is required for the anti-tumor efficacy in breast cancer of dual PI3K/mTOR inhibitors given in combination with MEK inhibitors
Authors: Smith Aleisha M. , Xiong Jessie , Hunter Lucas , Jordan Jamie , Clark Kelly , Darr David B. , Norman Sharpless , Perou Charles M. , Kim William Y.
Published: August 2015

Background: The mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways are deregulated in many cancers including breast cancer. Additionally, studies have shown that significant crosstalk exists between these pathways; often making single pathway inhibition ineffective. While, combined PI3K/mTOR and MEK inhibition appears highly active in pre-clinical models, the combination has been reported to be toxic in human use. In an effort to produce a more targeted regimen, we examined whether both PI3K and mTOR inhibition are required for pre-clinical anti-tumor efficacy in combination with a MEK inhibitor.

Methods: In vivo studies assessed overall survival and best tumor response upon administration of dual PI3K/mTOR (BEZ235 and GSK806) versus allosteric mTOR inhibition (Everolimus) in combination with MEK inhibition (AZD244 and GSK212) in genetically engineered murine models (GEMM) of breast cancer [T11 and C(3)Tag]. Activation and response of the PI3K/mTOR and MEK/ERK pathways to dual PI3K/mTOR, allosteric mTOR inhibition, and MEK inhibition were assessed in vitro in murine cell lines derived from the above GEM models.

Results: Single agent regimens of MEK, allosteric mTOR, or dual PI3K/mTOR inhibition were minimally effective in C(3)Tag or T11 tumors in vivo. As previously reported, combined PI3K/mTOR and MEK inhibition showed potent pre-clinical activity in both the T11 and C3Tag models, prolonging overall survival relative to both vehicle or single-agent treated mice. Surprisingly, combinations of MEK inhibitors with allosteric mTOR inhibitors were as active as the MEK/PI3K/mTOR combination. In vitro studies in cell lines derived from these tumor models showed that mTORC1 inhibition, but not PI3K inhibition, resulted in compensatory activation of MEK.

Conclusions: Combined targeting of the mTOR and MEK pathways results in improved response and survival in faithful GEM models of breast cancer. The combination of dual PI3K/mTOR catalytic inhibitors with MEK inhibition does not afford benefit over combined allosteric mTOR and MEK inhibition

Citation Format: Aleisha M. Smith, Jessie Xiong, Lucas Hunter, Jamie Jordan, Kelly Clark, David B. Darr, Sharpless Norman, Charles M. Perou, William Y. Kim. Inhibition of mTOR, but not PI3K, is required for the anti-tumor efficacy in breast cancer of dual PI3K/mTOR inhibitors given in combination with MEK inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2649. doi:10.1158/1538-7445.AM2015-2649
Abstract 2674: Activity of BAY1082439, a balanced PI3Kα/β inhibitor, in gastric cancer
Authors: Hung Huynh T. , Ong Richard , Haike Katja , Petrova Elissaveta , Chong Mei Ling , Loh Marie , Bhattacharya Bhaskar , Soong Richie , Liu Ningshu
Published: August 2015

Background: Activation of the p110α and p110β subunits of PI3K by PIK3CA mutation (7% of cancers), PIK3CA amplification (36-60%) and/or PTEN loss (33-47%) is a frequent oncogenic event in gastric cancer. Here, we report on the activity, mode of action and biomarker analysis of BAY1082439, a balanced PI3Kα/β inhibitor, in cell lines and xenografts from Asian and Caucasian gastric cancer patients.

Methods: Twenty cell lines were screened in vitro for the sensitivity to BAY1082439 using cell proliferation, cell cycle, migration and invasion assays. In vivo activity and mode of action of BAY 1082439 were further assessed by pathway inhibition, tumor growth inhibition, and effects on tumor stromal cells. Molecular features were assessed by Oncocarta analysis, gene expression arrays, and immunohistochemistry.

Results: Seven cell lines were sensitive to BAY1082439, which included 6/7 cell lines with PIK3CA mutations and/or PTEN loss. In vivo study with 15 PDX and 2 cell line models revealed that tumors with genetic alteration of PIK3CA and PIK3CB, PTEN-loss, and/or HER2/HER3/FGFR overexpression were more sensitive to BAY 1082439 (tumor growth inhibition >90%), while tumors harboring KRAS mutation and/or overexpressing EGFR were insensitive or less responsive to BAY 1082439. Interestingly, BAY1082439 did not inhibit proliferation of MKN45 cells in-vitro (IC50>10μM), however it induced tumor stasis in this highly vascularized MKN45 xenografts. The anti-angiogenic activity BAY 1082439 was also demonstrated in patient derived gastric cancer models assessed by CD31 immunohistochemistry. BAY1082439 was also active in many tumors resistant to standard-of-care chemotherapy. Synergistic or additive effects on tumor growth and apoptosis were observed for BAY1082439 in combination with paclitaxel, 5-FU, and capecitabine/oxaliplatin.

Conclusions: The PI3Kα/β balanced inhibitor BAY1082439 showed considerable anti-tumor activity including tumor regression in gastric cancer models with genetic alteration or activation of PI3Kα/β and/or PI3Kα/β as single agent or in combination.

Citation Format: Huynh T. Hung, Richard Ong, Katja Haike, Elissaveta Petrova, Mei Ling Chong, Marie Loh, Bhaskar Bhattacharya, Richie Soong, Ningshu Liu. Activity of BAY1082439, a balanced PI3Kα/β inhibitor, in gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2674. doi:10.1158/1538-7445.AM2015-2674
Abstract 2681: Stromal neuregulin-1 promotes adaptive resistance in mutant BRAF melanoma
Authors: Capparelli Claudia , Rosenbaum Sheera , Berger Adam , Aplin Andrew E.
Published: August 2015

RAF inhibitors are first-line treatments for melanomas that harbor BRAF V600E/K mutations; however, patients invariably progress on these targeted therapies. While durable resistance is mediated frequently by stable alterations intrinsic to the tumor cells leading to ERK1/2 pathway reactivation, the compensatory activation of growth factor receptor tyrosine kinases (RTKs) modulates the level of initial response. The composition of the tumor microenvironment likely plays a key role in these compensatory/adaptive responses to targeted inhibitors. In this study, we demonstrate that NRG1 is expressed in fibroblasts, as well as cancer associated fibroblasts (CAFs) isolated from a mutant BRAF melanoma. Fibroblast derived-NRG1 was able to mediate the enhanced ErbB3 pathway activation in RAF-inhibited mutant BRAF melanoma cells. Furthermore, conditioned medium from fibroblasts and CAFs limits RAF inhibitor cytotoxicity in V600 BRAF-harboring melanomas. Importantly, ErbB3 and ErbB2 targeting antibodies partially reverse the protective effects of fibroblast-derived medium on ErbB3 pathway activation and cell growth properties of mutant BRAF-inhibited melanoma cells. Together, these data demonstrate a requirement for a paracrine-produced factor in the tumor microenvironment to promote resistance to RAF inhibitor in mutant BRAF melanoma and reinforce the idea that targeting ErbB3/ErbB2 signaling may improve the efficacy of RAF inhibitors.

Citation Format: Claudia Capparelli, Sheera Rosenbaum, Adam Berger, Andrew E. Aplin. Stromal neuregulin-1 promotes adaptive resistance in mutant BRAF melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2681. doi:10.1158/1538-7445.AM2015-2681
Abstract 2683: Simultaneous inactivation of TP53 and loss of PTEN diminish response to targeted therapy in V600EBRAF mutant melanoma
Authors: Galvani Elena , Brooks Kelly , Baenke Franziska , Girotti Maria Romina , Gremel Gabriela , Kumar Mandal Amit , Viros Amaya , McManus Clare , Smith Matthew , Lim Kok Haw Jonathan , Lee Rebecca , Fusi Alberto , Lorigan Paul , Marais Richard
Published: August 2015

Numerous cancers are driven by mutationally activated kinases, many of which can be employed to define subsets of cancers and therefore patients most likely to benefit from treatment with rationally designed targeted therapies. Defining the landscape of genetic alterations that participate in cancer transformation provides insight into the diversity of clinical responses observed to targeted treatments. Here, we identified concurrent mutational inactivation of the tumor suppressors TP53 and PTEN as a mechanism of resistance to BRAF inhibitors in melanomas harboring mutant V600EBRAF.

Defects in TP53 function and PTEN loss occur in 17% and 12% of malignant melanomas respectively, rendering cells dependent on CHK1 to maintain normal cell cycle progression. We demonstrate that CHK1 inhibition increases sensitivity to a variety of alkylating agents and targeted therapies in V600EBRAF mutant, TP53/PTEN deficient cell lines and patient derived xenografts. Inhibition of CHK1 concurrently with the induction of either DNA damage or replication stress leads to ‘‘mitotic catastrophe’’ and cell death in our in vitro and in vivo models of TP53/PTEN deficient tumors. In the case of externally applied cellular stress from chemotherapeutics, our results suggest CHK1 inhibition may sensitise this genetically defined subset of melanomas to such treatments.

Our results provide some insight into the heterogeneity of clinical outcomes observed when treating BRAF mutant melanomas with BRAF inhibitor and suggest a need for comprehensive screening of TP53 and PTEN inactivation in these patients.

Citation Format: Elena Galvani, Kelly Brooks, Franziska Baenke, Maria Romina Girotti, Gabriela Gremel, Amit Kumar Mandal, Amaya Viros, Clare McManus, Matthew Smith, Kok Haw Jonathan Lim, Rebecca Lee, Alberto Fusi, Paul Lorigan, Richard Marais. Simultaneous inactivation of TP53 and loss of PTEN diminish response to targeted therapy in V600EBRAF mutant melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2683. doi:10.1158/1538-7445.AM2015-2683