bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2507: Circulating proteolytic products of carboxypeptidase N for early detection of breast cancer
Authors: Hu Ye
Published: May 2015

Background: Carboxypeptidase N (CPN) plays an important role in regulating vasoactive peptide hormones, growth factors, and cytokines by specifically cleaving their C-terminal basic residues. Herein we demonstrated that the circulating peptides specifically cleaved by CPN in the tumor microenvironment can indeed be stage-specific indicators of breast cancer.

Methods: The activity of CPN was evaluated using an ex-vivo peptide cleavage assay, in which synthesized C3f peptide (His6-C3f_S1304-R1320-His6) is incubated in interstitial fluids of breast tumor and adjacent normal breast tissues in mice with orthotopic implantation of the human cell line MDA-MB-231. Fragment profiling, by an approach combining nanopore fractionation and mass spectrometry, revealed the nature and extent of cleavage by CPN. These results correlated with the level of CPN-catalyzed peptides in blood specimens taken from the tumor-bearing mice, healthy women and breast cancer patients. CPN expression in the same set of samples was further examined by immunohistochemistry and immunoblotting.

Results: We showed that generation of C3f_R1310-L1319 specifically correlated with the CPN expression level. In both the mouse and clinical patient samples, the amount of CPN was clearly increased in tumor tissues compared to that seen in normal breast tissue, while its counterpart in blood remained constant. The amount of 6 CPN-catalyzed peptides predominantly increased in sera taken from the mice (n=8) at 2 weeks after orthotropic implantation. Six homologous peptides displayed the significantly higher expression in the patients' plasma as early as the first pathologic stage of breast cancer.

Conclusions: The amount of circulating CPN-catalyzed peptides reported here reflects the extent of this enzyme's activity in tumors, and our results clearly indicate their strong potential as biomarkers for non-invasive and early diagnosis of breast cancer. It would certainly be naïve to think that CPN-catalyzed peptides and the enzyme itself are the only biomarkers specific for breast cancer diagnosis. We do however argue that they can be used as a complementary measure for very early diagnosis of tumor growth, particularly because CPN activity within the tumor at this point is already detectable by blood sampling. Such sensitivity together with all of the features mentioned above lend well to their translation to the clinic, for breast cancer and perhaps other pathologies.

Citation Format: Ye Hu. Circulating proteolytic products of carboxypeptidase N for early detection of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2507. doi:10.1158/1538-7445.AM2014-2507
Abstract 2512: Design and antiproliferative evaluation of cell-penetrating TPM-Dox conjugates as prodrugs
Authors: Beni Yousef , Pippas Andrew W.
Published: May 2015

Doxorubicin (Dox) is well-known anthracycline widely used anticancer agent for the treatment of a wide variety of cancers. The major limitations of cancer chemotherapy treatment are low cellular uptake, high efflux rate and the development of resistance to a certain dose of anticancer drugs, such as Dox. Improving of cellular uptake and duration of action of Dox is very demanding. This will improve its therapeutic index and decrease its side effects. The biological activity of Dox can potentially be enhanced by increasing and optimizing its hydrophobicity using appropriate hydrophobic linker attachment to the Dox using triphenylmethanol (TPM) which led to higher cellular uptake. In this regard, TPM-Dox prodrugs were designed to deliver and release Dox into cells. The attachment of TPM analogs along with fatty alcohol significantly enhanced the lipophilicity and thus the cellular uptake of these multifunctional conjugates. In this study, a number of TPM derivatives of Dox were synthesized by the reaction of 2-substituted methoxy benzenes and 1,3,5-trioxane in glacial acetic acid in 72-87% yield. The reaction of TPM derivatives with tosylated fatty diol continued with conjugation to Dox to afford eight TPM-Dox conjugates with different hydrophobicity in 44-67% overall yield. Comparative antiproliferative assays between TPM-Dox conjugates and the corresponding noncovalent physical mixtures of the TPM derivatives and Dox were performed. TPM-Dox conjugates inhibited the cell proliferation of human leukemia (CCRF- CEM), ovarian adenocarcinoma (SK-OV-3), colorectal carcinoma (HCT-116), and breast carcinoma (MDA-MB-468) cells by 63-94% at a concentration of 1 µM after 72-120 h of incubation. These data suggest that TPM-Dox derivatives can be used as a potential prodrug for improving the cellular delivery and retention of Dox.

Citation Format: Yousef Beni, Andrew W. Pippas. Design and antiproliferative evaluation of cell-penetrating TPM-Dox conjugates as prodrugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2512. doi:10.1158/1538-7445.AM2014-2512
Abstract 2518: Development of a non-hydrolysable phosphotyrosine mimetic peptide based on a high affinity SHP2 substrate
Authors: Lawrence Harshani R. , Ge Yiyu , Becker Andreas , Ren Yuan , Luo Yunting , Wu Jie , Lawrence Nicholas J.
Published: May 2015

The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) is a transducer of growth factor, cytokine, integrin, and hormone signaling pathways that regulate processes such as cell proliferation, differentiation, adhesion, migration, and apoptosis and plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with several types of leukemias, and solid tumors. This makes SHP2 an attractive target for developing new anticancer therapy. Several small molecules have been developed for inhibition of this important phosphatase, which serve as chemical tools to probe the role of SHP2 in disease and lead compounds for optimization. Nevertheless improvements are required to improve these lead compounds with better potency, selectivity and cell permeability. To gain structural insights for development of potent and selective Shp2 inhibitors, we synthesized a non-hydrolysable phosphotyrosine peptide mimetic, based on reported Shp2 substrate peptide sequences. The non-hydrolyzable phosphopeptide, containing the phosphotyrosyl surrogate difluoromethylphosphonate, was prepared by solid phase peptide synthesis methods from the known building block N-FMOCF2Pmp. We will present the synthesis of the peptide, analysis of Shp2 phosphatase inhibition, and binding of the phosphopeptide mimic to SHP2 by micro isothermal calorimetry. We will present a model for the binding mode of the peptide and its implications for the design of selective Shp2 inhibitors.

Citation Format: Harshani R. Lawrence, Yiyu Ge, Andreas Becker, Yuan Ren, Yunting Luo, Jie Wu and Nicholas J. Lawrence. Development of a non-hydrolysable phosphotyrosine mimetic peptide based on a high affinity SHP2 substrate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2518. doi:10.1158/1538-7445.AM2014-2518
Abstract 2519: A small molecule that binds and inhibits the ETV1 transcription factor oncoprotein
Authors: Pop Marius , Stransky Nicolas , Garvie Colin , Theurillat Jean-Philippe , Lewis Timothy , Zhong Cheng , Culyba Elizabeth , Lin Fallon , Daniels Douglas , Pagliarini Raymond , Ronco Lucienne , Koehler Angela , Garraway Levi
Published: May 2015

Members of the ETS transcription factor family have been implicated in several cancers, where they are often dysregulated by genomic derangement. ETS variant 1 (ETV1) is an ETS factor gene that undergoes chromosomal translocation in prostate cancers and Ewing's sarcomas, amplification in melanomas, and lineage dysregulation in gastrointestinal stromal tumors. Pharmacologic perturbation of ETV1 would be appealing in these cancers; however, oncogenic transcription factors are often deemed “undruggable” by conventional methods. Here, we used small-molecule microarray (SMM) screens to identify and characterize drug-like compounds that modulate the biological function of ETV1. We identified the 1,3,5-triazine small molecule BRD32048 as a top candidate ETV1 perturbagen. BRD32048 binds ETV1 directly, modulating both ETV1-mediated transcriptional activity and invasion of ETV1-driven cancer cells. Moreover, BRD32048 inhibits p300-dependent acetylation of ETV1, thereby promoting its degradation. These results point to a new avenue for pharmacological ETV1 inhibition and may inform a general means to discover small molecule perturbagens of transcription factor oncoproteins.

Note: This abstract was not presented at the meeting.

Citation Format: Marius Pop, Nicolas Stransky, Colin Garvie, Jean-Philippe Theurillat, Timothy Lewis, Cheng Zhong, Elizabeth Culyba, Fallon Lin, Douglas Daniels, Raymond Pagliarini, Lucienne Ronco, Angela Koehler, Levi Garraway. A small molecule that binds and inhibits the ETV1 transcription factor oncoprotein. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2519. doi:10.1158/1538-7445.AM2014-2519
Abstract 2520: Novel inhibitors of β-catenin
Authors: Nurmemmedov Elmar , Cheltsov Anton , Vogt Peter K.
Published: May 2015

β-catenin is a key player of the WNT pathway; its aberrant regulation is associated with the onset and progression of numerous types of cancer. β-catenin is therefore an attractive drug target but direct inhibition of β-catenin is a challenging approach toward suppression of β-catenin-driven tumorigenesis. Ideal drug candidates should specifically target nuclear β-catenin, leaving its cytoplasmic and cell junctional functions intact. We have identified several drug-like candidate β-catenin inhibitors using a robust screening method against four allosteric binding sites of β-catenin (TCF4, BCL9 and two novel sites). We show that these inhibitors significantly reduce proliferation of a panel of human β-catenin-driven cancer cell lines, and repress the expression of β-catenin-induced target genes. They cause a shift in the melting temperature of the purified β-catenin protein. We also show that the candidate compounds reduce levels of β-catenin in the nucleus and block β-catenin-driven oncogenic transformation in cell culture. We consider these novel β-catenin inhibitors promising leads that should be further investigated for their molecular mechanism of action and for their potential development as therapeutic drugs.

Key words: β-catenin, oncogene, therapeutic, target, small molecule, protein-protein interaction

This work was supported by NIH Grant R01 CA078230. This is abstract 26043 of The Scripps Research Institute.

Citation Format: Elmar Nurmemmedov, Anton Cheltsov, Peter K. Vogt. Novel inhibitors of β-catenin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2520. doi:10.1158/1538-7445.AM2014-2520
Abstract 2523: Mechanism-based inhibitors of HGFA, matriptase and hepsin for breast cancer treatment
Authors: Janetka James , Han Zhenfu , Harris Peter , Wildman Scott
Published: May 2015

Hepatocyte Growth Factor Activator (HGFA) is a member of the S1 family of trypsin-like serine proteases. Its biological function is to proteolytically process the inactive single-chain zymogen precursors of the receptor tyrosine kinase ligands HGF (hepatocyte growth factor) and MSP (macrophage stimulating protein) resulting in formation of their active heterodimeric forms which initiate ligand-dependent c-MET and RON kinase cell signaling, respectively. We have discovered the first small molecular weight mechanism-based ketothiazole inhibitors of HGFA. The compounds were evaluated for HGFA, matriptase and hepsin inhibition in a protease competition assay using a chromogenic peptide substrate. We have identified several sub-micromolar inhibitors of HGFA and demonstrate they inhibit conversion of the native pro-HGF and pro-MSP substrates into active heterodimers. Finally, we show that HGFA inhibitors cause a dose-dependent decrease c-MET phosphorylation in invasive MDA-MB-231 breast cancer cells. This investigation provides preliminary validation of HGFA as a novel therapeutic target for breast cancer. These initial peptides serve as templates for lead optimization and the structure-based design of other small-molecule inhibitors. Optimized inhibitors will be used to study the role of HGFA in the regulation of growth factors, kinase receptor activation, cell signaling, invasion, and breast cancer metastasis.

Citation Format: James Janetka, Zhenfu Han, Peter Harris, Scott Wildman. Mechanism-based inhibitors of HGFA, matriptase and hepsin for breast cancer treatment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2523. doi:10.1158/1538-7445.AM2014-2523
Abstract 2525: Identification and characterization of class I HDAC-specific small-molecule inhibitors with a novel pharmacophore
Authors: Wang Yunfei , Stowe Ryan , Pinello Christie E. , Zhao Lisa Y. , Tian Guimei , Hodder Peter , Roush William R. , Liao Daiqing
Published: May 2015

Small-molecule inhibitors of HDACs (HDIs) have therapeutic potentials for treating cancer, metabolic disorders, cardiovascular diseases and neurodegenerative maladies. Two FDA-approved HDIs (vorinostat and romidepsin) are in clinical use for cancer therapy and a variety of other HDIs have been actively tested in clinical trials. Current challenges facing drug development of HDIs include the lack of isoform specificity, undesirable toxicity and suboptimal therapeutic efficacy against solid tumors. Most HDIs in preclinical or clinical evaluations belong to the hydroxamic acid or aminobenzamide classes. The former class lacks isoform specificity and the latter has not yet been approved for clinical use. Thus, the identification of HDIs with novel chemical properties and isoform-specificity may unleash the considerable therapeutic potential of targeting HDACs. We have conducted a high-throughput screening of >620,000 compounds and identified a lead with a hitherto undescribed pharmacophore with an inhibitory specificity against the class I HDACs, and a low two-digit nanomolar potency. An array of synthetic analogs established the structure-activity relationship. These novel inhibitors induced histone hyperacetylation, suppressed cancer cell proliferation of diverse cancer cell lines including the NCI-60 panel, and malignant cell-biological features associated with tumor metastasis. These compounds are under active preclinical development for cancer therapy.

Citation Format: Yunfei Wang, Ryan Stowe, Christie E. Pinello, Lisa Y. Zhao, Guimei Tian, Peter Hodder, William R. Roush, Daiqing Liao. Identification and characterization of class I HDAC-specific small-molecule inhibitors with a novel pharmacophore. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2525. doi:10.1158/1538-7445.AM2014-2525
Abstract 2532: Vemurafenib prodrugs suitable for oral and IV administration
Authors: Xi Ning , Zhang Yingjun , Wang Zhaohe , Lin Taoxi , Wang Qian
Published: May 2015

Background: The oral therapy vemurafenib was approved by the U.S. FDA in 2011 for first-line treatment of metastatic melanomas. Patients whose tumors carry the BRAF(V600E) mutation are treated with vemurafenib at recommended dose of 960mg twice daily. One reason for such a high dose is due to the poor oral bioavailability of vemurafenib. It was reported that the crystalline formulation of vemurafenib provided only modest drug exposures in animals and human (solubility limited absorption). To increase its oral bioavailability, vemurafenib was subsequently formulated as a micro-precipitated bulk powder (MBP). Despite this special formulation, vemurafenib displayed marked accumulation after repeat dosing at 960 mg BID with high inter-patient variability.

Methods and Results: To address the limitation of MBP formulation (e.g., high cost to manufacture and high doses to reach desired drug exposure), we developed prodrugs of vemurafenib, CT207 and CT317, with choices for both oral and i.v. administration. The prodrugs are homologues and freely soluble (>1g/mL) in water as their sodium salts. Both drugs are well-absorbed in dogs and monkeys when dosed in 100% saline solution or in capsule. For example, CT317 reached AUC(0-24) = 256 µMh and 429 µMh (calculated in vemurafenib form) when dosed to dogs at 30 and 50 mg/kg QD (equivalent to 1120 and 1890 mg/day adult dosage). The absolute oral bioavailability of CT207 and CT317 in dogs was consistent at ∼80% in dosing range of 10 to 100 mg/kg, which was approximately 4-times higher than vemurafenib (formulated as MBP). Excellent drug exposure was also observed when CT207 was given to monkeys orally at doses of 50 and 100 mg/kg QD (equivalent to 1120 and 2240 mg/day adult dosage), providing AUC(0-24) = 506 µMh and 955 µMh, respectively. Both CT207 and CT317 showed the same potent antitumor activity as vemurafenib in COLO205 xenograft tumor model, demonstrating the equivalency of tumoricidal effects of the prodrugs to vemurafenib.

Conclusions: The consistent high oral bioavailability across wide dosing range and great aqueous solubility of CT207 and CT317 render them to have many advantages over vemurafenib: choices for oral or intravenous administration, reduced daily dose and frequency, and minimized inter-patient variability, among others. Therefore, the prodrugs described here have the potential to complement the clinic use of vemurafenib.

Citation Format: Ning Xi, Yingjun Zhang, Zhaohe Wang, Taoxi Lin, Qian Wang. Vemurafenib prodrugs suitable for oral and IV administration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2532. doi:10.1158/1538-7445.AM2014-2532
Abstract 2541: Differential trafficking of dendritic cells after immunization determines establishment of systemic anti-tumor responses
Authors: Sato Noriko , Asiedu Kingsley , Choyke Peter L.
Published: May 2015

Systemic immune activation and memory cell generation is desired in dendritic cell (DC)-based therapies against cancers, especially with metastasis. Migration properties of DCs, however, could limit the responses regionally constrained. Clinically, DC-vaccines are often given subcutaneously (s.c.), intravenously (i.v.) or intradermally (i.d.), without monitoring the migration of DCs. In this study, we visualized trafficking of DCs labeled with In-111 oxine using a whole-body micro-SPECT imager after immunization via different routes and examined induction of immune activation and memory cell generation in mice. Bone marrow-derived DCs were used. Surprisingly, most of s.c. transferred DCs failed to migrate to the adjacent lymph nodes (LNs) and died at the injection site, irrespective of their maturation status. DCs injected i.v. were initially trapped in the lungs, but gradually migrated to the spleen, liver and, to a lesser extent, LNs. In contrast, DCs administered i.d. and intraperitoneally (i.p.) quickly entered lymphatic system and distributed to LNs. Proliferation of CMFDA labeled OT-1 CD8 T cells pre-transferred to mice receiving ovalbumin loaded DCs (OVA-DCs) indicated that i.v. and i.p. immunizations triggered stronger and more systemic expansion of effector T cells than s.c. and i.d. immunizations. To examine the establishment of long-term defense, we next immunized mice with OVA-DCs and challenged them with OVA-expressing tumors intramuscularly, contra-lateral to the s.c. and i.d. injection site. Intriguingly, i.v. immunization seemed less effective than the others in developing long-term antigen-specific immunity, despite its capability to induce an extensive acute responses. These results might suggest that difference in the microenvironment of organs where CD8 T cells encounter the antigen (i.e., spleen or LNs) controls the generation of long-lived memory cells. Collectively, immunization route would be a critical factor that determines the spatial distribution of immune activation and generation of functional memory cells in DC-based therapies.

Citation Format: Noriko Sato, Kingsley Asiedu, Peter L. Choyke. Differential trafficking of dendritic cells after immunization determines establishment of systemic anti-tumor responses. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2541. doi:10.1158/1538-7445.AM2014-2541
Abstract 2547: Peripheral immune signatures and survival in stage IV melanoma
Authors: Pawelec Graham P. , Martens Alexander , Garbe Claus , Weide Benjamin , Derhovanessian Evelyna , Schadendorf Dirk , Giacomo Anna Maria Di , Maio Michele , Vries Jolanda de , Zelba Henning
Published: May 2015

The presence of peripheral T-cells recognizing the melanoma-associated antigens NY-ESO-1 or Melan-A, as well as low frequencies of circulating CD14+ HLA-DR-/low myeloid-derived suppressor cells (MDSCs), is associated with prolonged survival in stage IV melanoma (n=94). In addition, the two of 12 patients with partial responses or stable disease in pilot studies of ipilimumab treatment both tended to have lower levels of MDSCs at baseline which decreased further on treatment. In contrast, two patients with progressive disease had high baseline MDSC levels, which increased further after treatment. These patients are being followed up to determine whether another mechanism of action of ipilimumab may be via an effect on MDSCs. A correlation was observed between the presence of antigen-reactive T cells and the frequency of MDSCs. Our results indicate that patients lacking antigen-reactive T cells had significantly higher levels of MDSCs. In addition, high frequencies of MDSCs correlated with lower frequencies of NY-ESO-1- and Melan-A-reactive T cells that also produced lower levels of IFN-gamma. NY-ESO-1 was recognized more frequently by CD4+ than by CD8+ T cells, whereas Melan-A more often stimulated CD8+ T cells. Possession of NY-ESO-1-reactive T cells conferred a survival advantage whether the response was mediated by CD4+ or CD8+ T cells or both. However, recognition of Melan-A by CD4+ T cells was associated with poorer clinical outcome even when the patient also possessed CD8+ reactive T cells. This negative effect was mediated by CD4+ T cells with a Th2- or Th17-type but not Th1-type cytokine response. Thus, tumor antigen-specificity and function of peripheral T cells and their interactions with MDSCs in advanced melanoma carries significant prognostic weight for predicting survival and may provide mechanistic insights into tumor immunosurveillance in such patients.

Citation Format: Graham P. Pawelec, Alexander Martens, Claus Garbe, Benjamin Weide, Evelyna Derhovanessian, Dirk Schadendorf, Anna Maria Di Giacomo, Michele Maio, Jolanda de Vries, Henning Zelba. Peripheral immune signatures and survival in stage IV melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2547. doi:10.1158/1538-7445.AM2014-2547
Abstract 2552: T cell apoptosis signaling biomarker of PD-1 engagement in cancer patients
Authors: Zhao Wei , Dong Haidong
Published: May 2015

Targeting membrane PD-1 molecules expressed by tumor-reactive T cells is gaining acceptance as a potentially effective anti-tumor immunotherapy. PD-1 blockade mediated by monoclonal antibodies has yielded promising therapeutic effects in treatment of melanoma, lung cancer and kidney cancers. Although detecting PD-1 expression by T cells is relatively straightforward, a simple and reliable method for determining whether PD-1 on T cells has engaged its ligand B7-H1 (PD-L1) is wanting. Our previous studies have revealed that engagement of PD-1 or B7-1 by B7-H1 results in alternations of apoptosis signaling pathway by up-regulating pro-apoptotic molecules among activated T cells and are correlated with B7-H1-mediated T cell death. Those observations suggest that intracellular levels of apoptosis-related Bcl2 family molecules among PD-1 positive CD8 T cells may be barometers of the extent to which PD-1 has been triggered by B7-H1. Using PD-1 and B7-H1 deficient animal models, we found that lower levels of intracellular pro-apoptosis molecules identify those activated T cells whose PD-1 molecules have not yet been extensively engaged, thus T cell function might be restored to their normal function once PD-1 signals from B7-H1 are blocked. Similarly, human PD-1 positive CD8 T cells engaged by B7-H1 also expressed higher levels of pro-apoptosis molecules during antigen stimulation. Our clinical data demonstrated that pro-apoptosis molecule expression by tumor-reactive T cell levels are higher among advanced melanoma and prostate cancers than healthy controls, reflecting chronic engagement with B7-H1. Thus, using pro-apoptosis status as signaling biomarkers for T cell PD-1 engagement, it may be possible to select patients who are most likely to have strong benefit from PD-1 or B7-H1 (PD-L1) blockade therapy.

Citation Format: Wei Zhao, Haidong Dong. T cell apoptosis signaling biomarker of PD-1 engagement in cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2552. doi:10.1158/1538-7445.AM2014-2552
Abstract 2555: A cell based bioluminescent reporter assay for rapid measuring function of PD-1 or PD-L1 therapeutic antibodies
Authors: Cong Mei , Karassina Natasha , Cheng Jey , Fan Frank
Published: May 2015

Programmed death-1(PD-1) and its ligand (PD-L1) are important immunotherapy targets for cancer. PD-1 serves as a negative costimulatory receptor on various cell types, including T and B cells as well as myeloid-derived cells. Its ligand PD-L1 (B7-H1) is not expressed by normal epithelial tissues, but it is aberrantly expressed on a wide array of human cancers including melanoma, non-small cell lung carcinoma, breast cancer, pancreatic cancer, etc. Engagement of PD-1 by its ligands PD-L1 expressed on tumor cells associated with poorer prognosis by disabling the host antitumor response. The most recent investigational anti-PD-1 immunotherapy drugs such as nivolumab from BMS and lambrolizumab (MK-3475) from Merck have all demonstrated significant overall survival rate in patients with advanced melanoma.

Here we developed a cell based bioluminescent reporter assay that can be used for rapid quantifying function of PD-1 or PD-L1 therapeutic antibodies as measured by activation of NFAT signaling pathway. For this, Jurkat T-cell line stably expressing NFAT-luciferase reporter and human PD-1 was generated. Raji stably expressing human PD-L1 was generated as antigen presenting cells (APC). by co-cultivating the two cell lines in the present of CD3 and IgG, Raji cells fully activate Jurkat NFAT pathway via CD28 binding to CD80 (B7-1) and CD86 (B7-2) endogenously expressed on Raji cells. PD-1 signaling in Jurkat cells following engagement of PD-L1 ectopically expressed on Raji cells inhibits T cell function, and results in NFAT pathway inhibition. Blockade of PD-L1 using anti-PD-L1 mAb reversed NFAT pathway activation.

Citation Format: Mei Cong, Natasha Karassina, Jey Cheng, Frank Fan. A cell based bioluminescent reporter assay for rapid measuring function of PD-1 or PD-L1 therapeutic antibodies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2555. doi:10.1158/1538-7445.AM2014-2555
Abstract 2566: Activation of tumor-initiated T cell priming and tumor destruction with potent STING-activating cyclic dinucleotide derivatives
Authors: Glickman Laura Hix , Kanne David B. , McWhirter Sarah M. , Leong Meredith L. , Lemmens Edward E. , Metchette Ken , Vance Russell E. , Pardoll Drew M. , Dubensky Thomas W.
Published: May 2015

Cyclic dinucleotides (CDNs) are bacterial messengers that are sensed by the host cytosolic surveillance pathway receptor, STING (Stimulator of Interferon Genes). CDN binding to STING initiates a TLR-independent signaling cascade through the TBK-1/IRF-3 axis, inducing type I interferon (IFN) and other co-regulated genes central to host innate defense. In response to binding cytosolic double-stranded DNA, a recently discovered mammalian enzyme, cyclic GMP-AMP synthase (cGAS), synthesizes CDNs that are structurally distinct from CDNs produced by bacteria. The mammalian CDN has a phosphate bridge with 2′-5′ and 3′-5′ linkages that increases its affinity for STING at least 10-fold. We show that mixed linkage (ML) CDNs were more potent activators of human PBMCs as compared to CDNs with canonical 3′-5′ phosphate linkages. Additionally, particular ML CDN derivatives with sulfur atoms at non-bridging oxygens of the internucleotide phosphate bridge were resistant to digestion by host cell phosphodiesterases, and R,R di-thio CDN diastereomers demonstrated increased potency in vitro and in vivo, as compared to either the R,S di-thio CDN diastereomer, or unmodified CDNs. Strikingly, direct injection of 2-week established flank B16 melanoma tumors with ML R,R dithio-CDN derivatives significantly and profoundly inhibited tumor growth, correlated with cytokine-mediated activation of NK and T lymphocytes, induction of antigen-specific T cell immunity and long-term survival. Anti-tumor efficacy was lost in mice encoding non-functional STING. Similar results were observed in other tumor models. These results demonstrate that CDNs have high translation potential for treatment strategies to induce activation of the tumor microenvironment, leading to effective tumor-initiated CD8+ T cell priming and anti-tumor efficacy.

Citation Format: Laura Hix Glickman, David B. Kanne, Sarah M. McWhirter, Meredith L. Leong, Edward E. Lemmens, Ken Metchette, Russell E. Vance, Drew M. Pardoll, Thomas W. Dubensky. Activation of tumor-initiated T cell priming and tumor destruction with potent STING-activating cyclic dinucleotide derivatives. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2566. doi:10.1158/1538-7445.AM2014-2566
Abstract 2568: Application of novel phospholipid conjugated Toll like receptor 7 ligands for cancer therapy by topical and systemic administration
Authors: Carson Dennis A. , Hayashi Tomoko , Crain Brian , Yao Shiyin , Cheng Jeffrey , Passini Nadia , Maj Roberto , Mura Emanuela , Cottam Howard , Holldack Johanna , Barberis Alcide
Published: May 2015

Introduction: Toll like receptor 7 (TLR7) activators are effective cancer immunotherapeutic drugs. However, the only clinically approved TLR7 ligand, imiquimod (IMQ), is absorbed into the systemic circulation inducing off-target adverse effects. To overcome this problem, we synthesized phospholipid-conjugated TLR7-specific activators. The objective of this project was to evaluate the safety and the anti-cancer activities of two TLR7 ligand-phospholipid conjugates, designated TMX-201 and TMX-202.

Procedures: In vitro immune activities of TLR7 agonists were compared in human and mouse leukocytes. Anti-malignant effects were evaluated using Inv-MycERTAM transgenic model of pre-malignant keratinocyte proliferation and murine models of 4T1 breast cancer and B16 melanoma.

Results: TMX-201 and TMX-202 were more potent cytokine inducers than IMQ in both mouse and human mononuclear cells. TMX-201 and TMX-202 showed less off-target binding compared to IMQ. The maximum tolerated doses of IMQ, TMX-201, and TMX-202 by i.v. administration were 15, 75 and 150 mg/kg, respectively. Topical administration of TMX-202 gel significantly reduced acanthosis in the Inv-mycERTAM model without inducing systemic cytokine production. Systemic administration of TMX-201 (4mg/kg) suppressed the growth of established subcutaneous B16 melanoma by 46%, without discernible adverse effects. Systemic or intra-tumor administration of TMX-201, in combination with anti-CTLA4 antibody, significantly suppressed lung metastasis formation in the 4T1 breast cancer model.

Conclusion: TMX-201 and TMX-202 displayed more potent immune stimulatory activity than IMQ with fewer off-target effects. These phospholipid-conjugated TLR7 agonists showed anti-cancer efficacy in three different mouse models. The new compounds may be useful topical and systemic therapeutic agents for cancer therapy, alone or in combination with checkpoint inhibitors.

Table 1.TMX-201 effect on lung metastases in the 4T1 breast cancer modelTreatmentMetastatic nodulesVehicle + isotype control33.5 ± 5.2TMX-20123.6 ± 4.9Anti-CTLA432.4 ± 13.6TMX-201 i.p. + anti-CTLA416 ± 2.7*TMX-201 i.t. + anti-CTLA413 ± 2.6**p<0.05 vs vehicle+isotype control group

Citation Format: Dennis A. Carson, Tomoko Hayashi, Brian Crain, Shiyin Yao, Jeffrey Cheng, Nadia Passini, Roberto Maj, Emanuela Mura, Howard Cottam, Johanna Holldack, Alcide Barberis. Application of novel phospholipid conjugated Toll like receptor 7 ligands for cancer therapy by topical and systemic administration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2568. doi:10.1158/1538-7445.AM2014-2568
Abstract 2572: CpG oligodeoxynucleotide enhances the efficacy of anticancer monoclonal antibody in an in vivo xenograft model using human endometrial cancer cell
Authors: Hiramatsu Kosuke , Serada Satoshi , Kobiyama Kouji , Morimoto Akiko , Kimura Toshihiro , Yoshino Kiyoshi , Fujimoto Minoru , Ishii Ken J. , Fujita Masami , Naka Tetsuji
Published: May 2015

Background: CpG oligodeoxynucleotides (ODN) has been shown to exhibit a potent immunostimulatory activity. Therefore, CpG ODN has been considered to be a promising material for enhancing the therapeutic effect of anticancer monoclonal antibody. We have previously identified bone marrow stromal antigen 2 (BST2) as a therapeutic target of endometrial cancer cells and also demonstrated that an anti-BST2 antibody showed a potent anti-tumor effect by antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against BST2 positive cells. The aim of this study was to evaluate the synergistic anti-tumor effect by combination of anti-BST-2 antibody and CpG ODN, and to clarify the mechanisms of its anti-tumor effect, using xenograft model of anti-BST2 positive endometrial cancer cells.

Material and Method: We implanted 7-8 week-old female SCID mice with 5×106 HEC-88 nu endometrial cancer cells subcutaneously. Mice with tumor were treated by intra-peritoneal injection of an anti-BST2 antibody alone (0 - 200 μg/mouse, twice a week for four weeks), intra-tumoral injection of CpG ODN alone (0 - 30 μg/mouse, twice a week for four weeks) or combination therapy (anti-BST2 antibody and CpG ODN). Anti-tumor effect was evaluated by monitoring the changes of tumor volume and also pathological analysis. In addition, activation of NK cells was evaluated.

Results: Intraperitoneal treatment of anti-BST2 antibody showed a significant anti-tumor effect, dose dependently. Similarly, intratumoral injection of CpG ODN showed a significant anti-tumor effect, in a dose dependent manner. Interestingly, the combination therapy was more effective than the single-agent therapy. In addition, the combination therapy also resulted in a high activity of NK cells.

Conclusion: We showed that CpG ODN increases the efficacy of anti-tumor effect of anti-BST2 antibody induced ADCC activity via potentiating NK cells mediated cancer cell injury. Combination of CpG ODN and anti-tumor monoclonal antibody is useful cancer therapy.

Citation Format: Kosuke Hiramatsu, Satoshi Serada, Kouji Kobiyama, Akiko Morimoto, Toshihiro Kimura, Kiyoshi Yoshino, Minoru Fujimoto, Ken J. Ishii, Masami Fujita, Tetsuji Naka. CpG oligodeoxynucleotide enhances the efficacy of anticancer monoclonal antibody in an in vivo xenograft model using human endometrial cancer cell. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2572. doi:10.1158/1538-7445.AM2014-2572
Abstract 2576: Polythymine oligonucleotides inhibit tumor growth through the induction of IFN alpha
Authors: Kobayashi Nobuaki , Shirota Hidekazu , Ichiki Yoko , Sakasai Kiyoshi , Ichikawa Yasushi , Kaneko Takeshi , Ishigatsubo Yoshiaki
Published: May 2015

IFNα is an immune-regulatory cytokine, used clinically for the treatment of various cancers. Administration of recombinant IFNα or IFNα-inducing agents in cancer patients induce the activation/expansion of NK cells and tumor-specific CTL responses, leading to tumor regression. Here, we find that the Polythymine (poly-T) oligonucleotides (ODN) induce the production of IFNα. Current results indicated the intratumoral injection of poly-T ODN could slow the growth of tumors and enhance the tumor specific CTLs. Our results shows a synthetic ODN containing Poly T motif have anti-tumor effect through the enhancement of IFNα production.

Citation Format: Nobuaki Kobayashi, Hidekazu Shirota, Yoko Ichiki, Kiyoshi Sakasai, Yasushi Ichikawa, Takeshi Kaneko, Yoshiaki Ishigatsubo. Polythymine oligonucleotides inhibit tumor growth through the induction of IFN alpha. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2576. doi:10.1158/1538-7445.AM2014-2576
Abstract 2581: Pancreatic cancer immunotherapy with chitosan/IL-12
Authors: Perlow Haley K. , Yang Lirong , Zaharoff David
Published: May 2015

Pancreatic cancer is difficult to treat due to its late diagnoses and limited treatment options. Strategies capable of reducing tumor mass so that patients are eligible for tumor resection, i.e. the Whipple procedure, and strategies for reducing metastatic deposits with immunotherapy are needed. New immunotherapy techniques can provide an alternative to modern chemotherapy, radiation, or surgical options which have not been able to reduce pancreatic cancer mortality. IL-12 has been discovered to be a potent agent in the development of anti-tumor immunity through its ability to stimulate the activities of cytotoxic T lymphocytes, NK cells, and macrophages while promoting Th1 differentiation. We have previously discovered that a bioadhesive chitosan/IL-12 combination can aggressively eliminate implanted tumors in murine models. Chitosan is a non-toxic and degradable polysaccharide derived from crustacean shells that shows great potential for use in immunotherapy models due to its unique binding properties. Here, ability of chitosan/IL-12 vaccinations to slow the growth of a pancreatic adenocarcinoma cells in vivo was investigated. C57BL/6 mice received subcutaneous inoculations with Panc02 cells. After Panc02 tumors became visible, the mice received 4 weekly intratumoral immunotherapy treatments with either saline or chitosan/IL-12. There was a prominent delay in the tumor growth of the chitosan/IL-12 group at all stages of the treatment. Seven days after the second treatment, control mice had an average tumor volume of 180mm3 and treatment mice tumors became undetectable. Seven days after the fourth treatment, the control mice had an average tumor volume of 743mm3 and treatment mice had an average tumor volume of 221mm3. Cell proliferation assays showed that IL-12 had no direct effect on the Panc02 cell line. However, interferon-γ, a cytokine whose production is induced by IL-12, reduced the growth of Panc02 cells in vitro. These data shows that chitosan/IL-12 may be an effective immunotherapy for pancreatic cancer and also that IL-12 is not directly cytotoxic, but rather, it initiates an effector response capable of inhibiting pancreatic tumors.

Citation Format: Haley K. Perlow, Lirong Yang, David Zaharoff. Pancreatic cancer immunotherapy with chitosan/IL-12. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2581. doi:10.1158/1538-7445.AM2014-2581
Abstract 2594: Adjuvant cationic nanoliposomes induce anti-cancer immunity in a murine model of breast cancer
Authors: Meraz Ismail M. , Savage David J. , Gu Jianhua , Rhudy Jessica , Serda Rita E.
Published: May 2015

Nanoparticles, such as liposomes, provide opportunities to simultaneously present antigens and immune modulators. Using a 4T1 murine model of breast cancer, a cationic nanoliposomal formulation containing monophosphoryl lipid A and the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane induced anti-tumor activity following intratumoral administration. Addition of recombinant interleukin-12 (IL-12) further suppressed tumor growth and augmented T helper-1 cell (Th-1) polarization, with enhanced tumor infiltration by cytotoxic T cells, dendritic cells, and M1 macrophages, and amplification of interferon gamma secretion. Mice bearing dual tumors displayed arrest of tumor growth in treated tumors as well as distal, untreated tumors following combination therapy with adjuvant nanoliposomes and IL-12. In summary, adjuvant MPL-liposomes combined with localized IL-12 therapy block tumor growth, stimulate a Th-1 bias of the tumor microenvironment, and induce cancer-specific immune responses.

Citation Format: Ismail M. Meraz, David J. Savage, Jianhua Gu, Jessica Rhudy, Rita E. Serda. Adjuvant cationic nanoliposomes induce anti-cancer immunity in a murine model of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2594. doi:10.1158/1538-7445.AM2014-2594
Abstract 2597: Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices
Authors: Leshchiner Elizaveta , Bellairs Joseph , Bird Gregory H. , Opoku-Nsiah Kwadwo , Godes Marina , Walensky Lored D.
Published: May 2015

Activating mutations in KRAS represent the most frequent oncogenic driving force among the RAS homologs K-, N- and H-RAS, and are associated with poor prognosis and chemoresistance. KRAS mutations are present in approximately 30% of tumors, and at even higher frequencies in cancers of the pancreas, lung, thyroid gland, colon, and liver. In pancreatic ductal adenocarcinomas (PDAC), which carries a 5-year survival rate of less than 5%, activating KRAS mutations are present in over 90% of tumors. Moreover, these mutations have been causally linked to the initiation and progression of PDAC. Despite the mechanistic insights into KRAS-mediated oncogenesis, development of high affinity targeted inhibitors remains a formidable challenge. Here, we report the synthesis and application of hydrocarbon-stapled peptides as prototype therapeutics for blocking wild type and mutant KRAS in vitro and in cancer cells. Stabilized Alpha-Helices of SOS1 (SAH-SOS1) were generated by inserting hydrocarbon staples into SOS1 peptide sequence to recapitulate the alpha-helical structure of the native KRAS-interaction domain. SAH-SOS1 peptides, but not negative control analogs, bound to a variety of KRAS constructs with nanomolar affinity, inhibiting nucleotide exchange. This sequence-specific biochemical activity correlated with impairment of KRAS-driven cancer cell viability and signal transduction. These studies provide proof-of-concept for the utility of SAH-SOS1 peptides in dissecting and targeting the oncogenic KRAS pathway in human cancer.

Citation Format: Elizaveta Leshchiner, Joseph Bellairs, Gregory H. Bird, Kwadwo Opoku-Nsiah, Marina Godes, Lored D. Walensky. Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2597. doi:10.1158/1538-7445.AM2014-2597
Abstract 2599: KDM3A tyrosine phosphorylation by Ack1 promotes tamoxifen-resistance in breast cancer
Authors: Mahajan Kiran , Bandyopadhyay Sayantani , Mahajan Nupam
Published: May 2015

Hormone therapy with the selective estrogen-receptor modulator tamoxifen (TAM) provides palliative benefit for patients with breast cancer. However, estrogen receptor α (ER hereafter) positive breast tumors with high expression of HER2 receptor tyrosine kinase often develop significant resistance to tamoxifen therapy. Thus, precise understanding of activation of Estrogen (E2)-independent ER regulated gene transcription in tamoxifen-resistant breast tumors could open new therapeutic avenues to target drug-resistance and ameliorate poor prognosis. The non-receptor tyrosine kinase, Ack1 (also known as TNK2) has emerged as a major integrator of signaling from various receptor tyrosine kinases including HER2. Here we demonstrate that heregulin promoted ER activity at Estrogen Response Element (ERE) in the presence of tamoxifen, which was significantly downregulated upon Ack1 knockdown. We observed that Ack1 phosphorylated the ER co-activator, KDM3A, a H3K9 demethylase, at evolutionary conserved tyrosine 1114 in heregulin dependent manner in the presence of tamoxifen. KDM3A demethylates H3 mono- and dimethyl-K9 in vitro and in vivo. Heregulin mediated Ack1 activation resulted in significant decrease in the level of monomethyl-H3K9, while, monomethyl-H3K27 levels remained unchanged. Further, inhibition of Ack1 by small molecule inhibitor AIM-100 or Dasatinib resulted in significant increase in the level of monomethyl-H3K9. Therefore by its ability to regulate the activity of ER co-activator such as KDM3A, activated Ack1 may modulate ER target gene expression in the absence of estrogen and confer TAM-resistance to cancer cells. Thus, Ack1 inhibitors hold the potential as novel therapeutics to treat a subset of tamoxifen resistant breast cancers.

Citation Format: Kiran Mahajan, Sayantani Bandyopadhyay, Nupam Mahajan. KDM3A tyrosine phosphorylation by Ack1 promotes tamoxifen-resistance in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2599. doi:10.1158/1538-7445.AM2014-2599