bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5440: Novel PD-1 blockade bioassay to assess therapeutic antibodies in PD-1 and PD-L1 immunotherapy programs
Authors: Cheng Zhi-Jie Jey , Karassina Natasha , Grailer Jamison , Hartnett Jim , Fan Frank , Cong Mei
Published: August 2015

Programmed death receptor-1 (PD-1) and its ligand (PD-L1) are among the few important immunotherapy targets for cancer. Current PD1 assays measure cell proliferation or cytokine production in primary T cells which are tedious, have high assay variation and small assay window. To enable quantitative potency measurement for key anti-PD-1 drugs in the market or in clinical trials such as pembrolizumab and nivolumab, as well as anti-PD-L1 drugs in clinical trials such as MPDL3280A and BMS-936559, here we report the development of a robust bioluminescent cell-based PD1 blockade bioassay. For this, we built a PD-1 effector cells in Jurkat cells which stably express human PD-1 and a NFAT-RE-luciferase reporter, and a PD-L1 positive artificial Antigen Presenting Cells (PD-L1+ aAPC) in CHO-K1 cells which stably express PD-L1 and an engineered TCR activator. Once these two cell types were co-cultivated, transcriptional activation of NFAT pathway in PD-1 effector cells, mediated by binding of TCR complex with TCR activator in PD-L1+ aAPC, is significantly suppressed by PD-1/PD-L1 engagement. This inhibition can then be specifically reversed by co-incubation of PD-1 or PD-L1 blocking antibodies in dose-dependent manner, but not by the antibody for other immune checkpoint receptors such as anti-CTLA4 ipilimumab. We further developed both PD-1 effector cells and PD-L1+ aAPC in Thaw-and-Use format so the cells can be plated for assay without the need of cell culture. The resultant PD-1 assay using Thaw-and-Use cells brings the benefit of convenience, low day-to-day variation, and easy lab-to-lab assay transfer. We demonstrate the assay is able to measure relative potency for antibody biologics, and also can detect potency changes for stressed antibody samples. In summary, the reporter-based PD-1 blockade assay provides a valuable tool for both drug screening and characterization in early drug discovery, and lot release and stability study in drug manufacture for therapeutic antibody drug candidates in PD-1 and PD-L1 immunotherapy programs.

Citation Format: Zhi-Jie Jey Cheng, Natasha Karassina, Jamison Grailer, Jim Hartnett, Frank Fan, Mei Cong. Novel PD-1 blockade bioassay to assess therapeutic antibodies in PD-1 and PD-L1 immunotherapy programs. [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 5440. doi:10.1158/1538-7445.AM2015-5440
Abstract 5455: Surface accumulation of receptor tyrosine kinases upon treatment with tyrosine kinase inhibitors and resulting enhancement of activity upon treatment cessation
Authors: Bjerregaard Anne-Mette , Grandal Michael V. , Frohlich Camilla , Lindsted Trine , Egebjerg Christina , Horac Ivan D. , Kragh Michael , Pedersen Mikkel W.
Published: August 2015

Receptor tyrosine kinases (RTKs) constitute the largest family of structurally related oncogenes and are often involved in the initiation, maintenance and/or metastasis of various human cancers. The genes encoding RTKs are frequently amplified, fused, mutated, or the ligands are expressed at high levels, causing abnormal activation and downstream signaling. Two major types of drugs have been used with success to treat cancers with abnormal RTK activation, recombinant monoclonal antibodies (mAbs) and small molecule inhibitors of the tyrosine kinases (TKIs). TKIs have proven most successful against cancers driven by gene fusions or mutations, whereas mAbs dominate in cancers driven by gene amplifications or ligand-mediated RTK activation. Recent studies show that the two types of treatment modalities may often be synergistic, but the reason for this is not well characterized.

Here, we demonstrate that surface levels of various RTKs increase in cancer cell lines upon treatment with TKIs specific for the individual RTKs. The effect was most pronounced in cell lines with amplification or autocrine ligand production and occurred at inhibitor concentrations suboptimal for inhibition of growth. Removal of the TKIs resulted in an increase in RTK activity and cancer cell proliferation as a result of the increased RTK levels.

MAbs, especially mAb mixtures, when given in combination with the TKIs were found to prevent RTK accumulation and led to enhanced efficacy. Due to the increased number of receptors, the combination also resulted in enhancement of secondary effector functions, such as ADCC.

In conclusion, our results demonstrate that in certain contexts TKI treatment leads to RTK accumulation and that this accumulation has the potential to enhance tumor growth. Antibody mixtures, when given together with the TKIs, can prevent RTK accumulation and even enhance the TKI activity.

Citation Format: Anne-Mette Bjerregaard, Michael V. Grandal, Camilla Frohlich, Trine Lindsted, Christina Egebjerg, Ivan D. Horac, Michael Kragh, Mikkel W. Pedersen. Surface accumulation of receptor tyrosine kinases upon treatment with tyrosine kinase inhibitors and resulting enhancement of activity upon treatment cessation. [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 5455. doi:10.1158/1538-7445.AM2015-5455
Abstract 5456: FGF9 gene amplification can induce resistance to anti-EGFR therapy in colorectal cancer
Authors: Mizukami Takuro , Togashi Yosuke , Banno Eri , Terashima Masato , de velasco Marco A , Sakai Kazuko , Fujita Yoshihiko , Tomida Shuta , Eguchi Nakajima Takako , Boku Narikazu , Nishio Kazuto
Published: August 2015

The prognosis of advanced colorectal cancer (CRC) remains dismal. Fibroblast growth factor (FGF) signals are potent mitogens, morphogens, and inducers of angiogenesis. Abnormal expression of FGF9 has been observed in several malignancies and might result in uncontrolled cell proliferation and carcinogenesis. However, limited information is available regarding the role of FGF9 in CRC. In this study, amplification of FGF9 gene was observed in 14/146 (9.6%) CRC specimens, using a TaqMan copy number assay. FGF9-overexpressed CRC cell lines were created, and FGF9-overexpression induced the resistance to anti-epidermal growth factor receptor (EGFR) therapy (cetuximab or lapatinib) via the FGF receptor (FGFR) signal, which was cancelled by BGJ398, a pan-FGFR inhibitor. In vivo, FGF9-overexpressed xenograft tumors were resistant to cetuximab monotherapy and the combination with BGJ398 overcame the resistance. Considering these results, FGF9 gene amplification may play an important role in the resistance to anti-EGFR therapy in CRC and the combination with an anti-FGFR inhibitor could overcome this resistance.

Citation Format: Takuro Mizukami, Yosuke Togashi, Eri Banno, Masato Terashima, Marco A de velasco, Kazuko Sakai, Yoshihiko Fujita, Shuta Tomida, Takako Eguchi Nakajima, Narikazu Boku, Kazuto Nishio. FGF9 gene amplification can induce resistance to anti-EGFR therapy in colorectal 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 5456. doi:10.1158/1538-7445.AM2015-5456
Abstract 5457: MAD2γ, a new MAD2 isoform, is ubiquitously expressed in distinct cell lines, reduces mitotic arrest, and associates with resistance to cisplatin-based chemotherapy in testicular germ cell tumors
Authors: Lopez-Saavedra Alejandro , Caceres Rodrigo , Luna Fernando , Hernandez Irwin , Herrera Luis Alonso
Published: August 2015

The mitotic spindle assembly checkpoint (SAC) signaling has been emerging as an important promising mechanism in cancer therapy. A prolonged mitotic arrest in response to cancer chemotherapy induces apoptosis, mitotic catastrophe, mitotic slippage and senescence, depending on what cell line and/or inhibitor is studied. In consequence, a weakened or disturbed SAC signaling in human cancers might contribute to drug resistance both to spindle inhibitors and to DNA damaging agents. In several studies the mitotic arrest deficient 2 (MAD2α) has been implicated in the association between failure in the SAC signaling and chemotherapy response. In this study, we identified a novel splicing variant of MAD2, designated as MAD2γ. This isoform was ubiquitously expressed in several cancer cell lines and non-cancerous primary foreskin fibroblasts. When it was ectopically expressed, MAD2γ localized in the nucleus. Its overexpression in a fully functional SAC-competent cancer cell line, HCT116, reduced the mitotic index, suggesting SAC impairment. Furthermore, the endogenous overexpression of MAD2γ in testicular germ cell tumor patients was associated with a resistant response to cisplatin-based chemotherapy, whereas expression of MAD2α was not significantly different between resistant and sensitive patients. In addition, HCT116 raised significantly its endogenous expression of MAD2γ only in response to cisplatin, but not that of MAD2α. Our data suggest that MAD2γ may have an opposing role to MAD2α in the activation of the SAC signaling, and that its expression is associated to a resistant response to DNA-damaging agents, such as cisplatin-based chemotherapy. This highlight the importance to study alternative splicing that may compromise the SAC function, which has been regarded as a promise for cancer therapy.

Citation Format: Alejandro Lopez-Saavedra, Rodrigo Caceres, Fernando Luna, Irwin Hernandez, Luis Alonso Herrera. MAD2γ, a new MAD2 isoform, is ubiquitously expressed in distinct cell lines, reduces mitotic arrest, and associates with resistance to cisplatin-based chemotherapy in testicular germ cell tumors. [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 5457. doi:10.1158/1538-7445.AM2015-5457
Abstract 5458: Elevated level of Fibrillarin induces chemoresistance by interfering p53 pathway in cancers
Authors: Xu Teng , Ganapathy Suthakar , Long Meijun , Ha Chul , Yuan Zhi-Min , Su Hang
Published: August 2015

Fibrillarin (FBL), an rRNA 2′-O-methyltransferase, has long been recognized as the enzymatic unit of the C/D box small nucleolar ribonucleoprotein (snoRNP) involved in the first step of pre-rRNA processing. It is not until recently that the link between FBL and cancers has been unveiled. As a direct regulatory target of Myc and p53, FBL is frequently upregulated in breast cancers and prostate cancers. High expression of FBL has also been shown to be associated with poor survival in patients with breast cancer. Our recent study found that FBL overexpression contributes to tumorigenesis and confers cellular resistance to chemodrugs. However, the underlying mechanism of chemoresistance elicited by FBL upregulation is still elusive. Here, we studied the molecular roles of FBL on p53 response. The p53 pathway in response to etoposide and actinomycin D were examined in a variety cell lines including U-2OS, MCF7, H1299, ZR75-1, A549 and an FBL inducible cell line. Western blotting and immunofluorescent staining were employed to examine protein level and their cellular localization. We found that ectopic expression of FBL enhanced the protein level of MDM2, resulting in decreased p53 accumulation and activity in response to chemodrugs. Interestingly, MDM2 was partially recruited to the nucleoli under stimuli. FBL depletion abrogated the nucleolar recruitment of MDM2 and subsequently dampened p53 activation. In conclusion, physiological level of FBL sequesters and stabilizes MDM2 in the nucleoli, therefore inhibits p53 degradation. When upregulated in cancers, FBL stabilizes nucleus MDM2 and promotes the degradation of p53

Citation Format: Teng Xu, Suthakar Ganapathy, Meijun Long, Chul Ha, Zhi-Min Yuan, Hang Su. Elevated level of Fibrillarin induces chemoresistance by interfering p53 pathway in 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 5458. doi:10.1158/1538-7445.AM2015-5458
Abstract 546: Soluble EphA2 is a potential serum biomarker for lung cancer
Authors: Kondo Tadanori , Yumoto Hiroki , Usui Kento , Kato Kazunori
Published: August 2015

EphA2 is a cell surface glycoprotein expresses in various tumor tissues including lung, breast and pancreas carcinoma. As the patients with a highly positive surface expression of EphA2 carcinomas have the poor prognosis, therefore, preclinical and clinical studies using antibody targeting to EphA2 are underlying. Previous study has reported that EphA2 fragment is also released in a soluble form by the proteolytic cleavage from tumor cell surface. In this study, we tried to detect the soluble form of EphA2 (sEphA2) in the serum specimens of lung cancer patients. By using antibodies to EphA2, we developed a new sandwich ELISA system detecting human sEphA2 with high sensitivity and specificity. With this system, we measured the concentration of sEphA2 in the serum of normal healthy donors and lung cancer patients. The concentration of sEphA2 was significantly higher in sera of 15 lung cancer patients than 15 healthy donors (p<0.01). We also examined the other diagnostics biomarkers such as CYFRA and CEA. The levels of sEphA2 in serum of lung cancer patients are correlated with CYFRA and CEA. These data indicate that the diagnosis of the lung cancer enabled by measuring the concentration of sEphA2. However, detail clinicopathological characteristics are necessary for the feasible diagnosis for sEphA2 as a biomarker of lung cancer.

Citation Format: Tadanori Kondo, Hiroki Yumoto, Kento Usui, Kazunori Kato. Soluble EphA2 is a potential serum biomarker for lung 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 546. doi:10.1158/1538-7445.AM2015-546
Abstract 5462: Extracellular ATP promotes cancer cell drug resistance to tyrosine kinase inhibitors by competing for the ATP-binding site of tyrosine kinase
Authors: Wang Xuan , Qian Yanrong , Li Yunsheng , Chen Xiaozhuo
Published: August 2015

ATP plays important roles in cellular processes in cancer cells, which are a heterogeneous population in tumors regarding their genetic background and oxygen and energy supplies. The fact that cancer cells undergo rapid proliferation suggests that they must possess mechanisms, which may not be present in normal cells, to meet their high energy needs. Studies by others revealed that extracellular ATP levels of various cancer types are 10^3 to 10^4 times higher than those in their corresponding normal tissues (1). We recently reported that extracellular ATP can be taken up directly by cancer cells by macropinocytosis (2). Based on these results, we hypothesized that the internalized extracellular ATP not only promotes cancer cell growth and survival, but also drug resistance. In this study, human lung cancer A549 and breast cancer MCF7 cells were used as cell models to determine the effects of extracellular ATP on drug resistance to tyrosine kinase inhibitors (TKIs) in cancer cells. Cancer cell viability and intracellular ATP measurement results showed that extracellular ATP substantially increased intracellular ATP levels and promoted cancer cell drug resistance to TKIs. Viability of TKI treated cancer cells was significantly increased in the presence of extracellular ATP, compared to cancer cells with TKI treatment alone. Administration of macropinocytosis inhibitors significantly reduced the drug resistance induced by extracellular ATP, suggesting that extracellular ATP promoted drug resistance by contributing to the intracellular ATP pool. Protein and protein phosphorylation analyses results suggested that the increased intracellular ATP reversed the inhibition of TKIs by competing with the inhibitor for the ATP-binding site of platelet-derived growth factor receptor (PDGFR) and enhancing Akt and MAPK signaling pathways. These findings identify, for the first time, new biological functions of extracellular ATP in tumors and may lead to novel anti-cancer strategies combating drug resistance in cancers.

1. Pellegatti P, et al. PLoS ONE 2008;3:e2599.

2. Qian Y, et al. Cancer Lett 2014;351:242-51.

Citation Format: Xuan Wang, Yanrong Qian, Yunsheng Li, Xiaozhuo Chen. Extracellular ATP promotes cancer cell drug resistance to tyrosine kinase inhibitors by competing for the ATP-binding site of tyrosine kinase. [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 5462. doi:10.1158/1538-7445.AM2015-5462
Abstract 5465: Systematic interrogation of druggable pathways in pancreatic adenocarcinomas using pooled gene-knockdown lentiviral libraries
Authors: Ferrer-Lorenzo Edgar , Nussbaum Daniel P. , Winter Peter , Wood Kris
Published: August 2015

Despite significant advances in the development of cancer treatment in the last 20 years the best available therapies have marginally improved the survival rate of treated pancreatic cancer patients (from 3% to 6% five-year survival), making it still one the deadliest malignancies. Single standard of care cytotoxic and targeted therapies demonstrate little to no effect on patient survival. Even though we now understand some of the main genetic factors that are mutatated in pancreatic tumors, their role in drug response remains poorly understood. To identify drug-sensitizing genetic targets in cancer cells we have developed a high-throughput screening platform. In this methodology, pooled lentiviral libraries containing hundreds of knockout constructs specific for cancer-related genes are used to infect cancer cells. By treating the infected populations with therapeutic agents we are able to identify genetic targets that when inhibited synergize with existing drugs to cause increased cell death. We piloted this type of screening platform in HCT-116 cells, a KRAS mutant, MEK inhibitor resistant colorectal cancer cell line, using the MEK-inhibitor AZD-2644 and a pooled shRNA library. This study identified 4-7 target genes that when knocked down sensitized the previously resistant HCT-116 cells to AZD-2644. To conduct a more robust and representative sensitization screen we developed a lentiviral CRISPR-Cas9 library. By performing screens in a panel of pancreatic cancer cell lines, we have identified both cell line dependencies and sensitizers to targeted and cytotoxic chemotherapies. The findings from these screens provide potential mechanistic insights into the key survival signaling programs in pancreatic cancer as well as new therapeutic strategies.

Citation Format: Edgar Ferrer-Lorenzo, Daniel P. Nussbaum, Peter Winter, Kris Wood. Systematic interrogation of druggable pathways in pancreatic adenocarcinomas using pooled gene-knockdown lentiviral libraries. [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 5465. doi:10.1158/1538-7445.AM2015-5465
Abstract 5473: Human cancer cells acquire chemoresistance to gemcitabine mainly through loss-of-function mutations in the DCK gene
Authors: Horii Akira , Saiki Yuriko , Nakano Tomohiro , Kudo Chiharu , Sunamura Makoto
Published: August 2015

Gemcitabine (GEM) has shown clinical activity in several solid tumors such as pancreatic and biliary tract cancers, urinary cancer, and non-small cell lung cancers, but a substantial number of patients acquire GEM chemoresistance. GEM functions after phosphorylation by deoxycytidine kinase (DCK) and its inactivation is one of the important mechanisms for acquisition of gemcitabine resistance. We established eight GEM resistant cell lines including four pancreatic, two biliary tract, and two gastrointestinal cancers and investigated mechanisms for acquisition of GEM resistances; six GEM resistant cell lines harbored complete inactivating mutations of DCK by protein-truncating mutation or complete or partial loss of the gene, and one gastric cancer cell line harbored a missense mutation that inactivate DCK activity. One pancreatic cancer cell line lost GEM sensitivity by unknown mechanism. Our present results strongly suggest that genetic alteration of DCK can be the major cause of acquisition of GEM resistance. Further studies analyzing primary tumors will give us (1) the fully understanding of acquisition of GEM resistance, and (2) desirable clues to overcome the acquisition of GEM resistance.

Citation Format: Akira Horii, Yuriko Saiki, Tomohiro Nakano, Chiharu Kudo, Makoto Sunamura. Human cancer cells acquire chemoresistance to gemcitabine mainly through loss-of-function mutations in the DCK gene. [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 5473. doi:10.1158/1538-7445.AM2015-5473
Abstract 5477: MERIT40 is an Akt substrate that promotes resolution of DNA damage induced by chemotherapy
Authors: Brown Kristin K. , Montaser-Kouhsari Laleh , Beck Andrew H. , Toker Alex
Published: August 2015

Resistance to cytotoxic chemotherapy agents is a common phenomenon in breast cancer and has a drastic impact on patient survival. Characterization of the mechanisms that contribute to chemotherapy resistance, and identification of strategies to circumvent resistance, would provide more effective therapies for the management of breast cancer. In this study we show that in addition to inflicting DNA damage, the cytotoxic chemotherapy agent doxorubicin triggers rapid and sustained activation of Akt in breast cancer cells. Pharmacological inhibitors of PI 3-K or Akt effectively block Akt signaling downstream of DNA damage and render cells markedly more sensitive to doxorubicin. We sought to identify mechanisms by which Akt promotes resistance to doxorubicin and have identified MERIT40, a component of the nuclear BRCA1-A DNA damage repair complex, as a novel Akt substrate that is phosphorylated when cells are exposed to doxorubicin. We have demonstrated that MERIT40 phosphorylation is necessary for BRCA1-A complex assembly and is required for resolution of DNA damage. We propose that inhibition of MERIT40 phosphorylation contributes to the efficacy of combination therapy with PI 3-K or Akt inhibitors and doxorubicin. We also show that MERIT40 phosphorylation is associated with ER status in breast tumor tissue samples, suggesting that MERIT40 phosphorylation could be used as a biomarker in ER-positive breast cancer patients to predict sensitivity to cytotoxic chemotherapy agents. Our data also indicates that patients with high levels of phosphorylated MERIT40 would likely benefit from combination therapy with Akt inhibitors and doxorubicin. We are currently investigating additional mechanisms by which Akt activation downstream of DNA damage promotes chemotherapy resistance with the aim of identifying novel combination therapy regimens to circumvent chemotherapy resistance.

Citation Format: Kristin K. Brown, Laleh Montaser-Kouhsari, Andrew H. Beck, Alex Toker. MERIT40 is an Akt substrate that promotes resolution of DNA damage induced by chemotherapy. [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 5477. doi:10.1158/1538-7445.AM2015-5477
Abstract 5487: Functional analysis of rare dihydropyrimidine dehydrogenase variants relevant to 5-fluorouracil toxicity
Authors: Shrestha Shikshya , Offer Steve , Diasio Robert B.
Published: August 2015

5-Fluorouracil (5-FU) is a cornerstone drug for the treatment of several aggressive tumors; however, adverse 5-FU toxicity contributes to approximately 1,300 deaths annually in the U.S. While three genetic variations in dihydropyrimidine dehydrogenase (DPD; DPYD gene) are well-established predictors of toxicity (i.e., *2A, I560S, D949V), the contributions of additional reported missense DPYD variants (>130) have not been adequately evaluated in clinical studies. Our lab has previously developed methods to study DPYD variants for potential clinical relevance to 5-FU toxicity using in vitro and cellular approaches (see Offer et al. Cancer Research 2013 & 2014). In the current study, we measured the in vitro enzyme activity of 47 uncharacterized missense variants that have been previously reported in dbSNP and NHLBI ESP databases. Additionally, we assessed the impact of these variants on 5-FU sensitivity in multiple transgenic human cellular models using real-time cellular analysis. Approximately one-third of the studied variants showed significantly reduced enzyme activity compared to wild type. Of the DPD-deficient variants identified in this study, 8 were profoundly deficient (<12.5% of “wildtype” activity), with enzyme activities similar to *2A. Two deficient variants showed modest reductions in enzyme activity (12.5-25% activity), similar to I560S. The remaining 5 deleterious variants exhibited mild deficiency (>25% activity, but significantly lower than “wildtype”), similar to D949V. Cellular sensitivity to 5-FU for cells expressing these variants paralleled the in vitro enzyme activity results, with deleterious variants showing reduced tolerance to 5-FU relative to cells transgenic for wildtype DPD. These trends were observed in multiple cell lines derived from various tissues and tumor types. Overall, our results add to the growing database of deleterious DPYD variations. Many of these variants have been observed primarily in non-Caucasian racial groups, consistent with our previous reports. These data suggest that the deleterious variants classified in this study may be important predictors of 5-FU toxicity, particularly in individuals of non-Caucasian ancestry. Additionally, these data suggest that genetic testing for rare variants may improve the success of predictive tests for 5-FU toxicity. Finally, this study suggests that in vitro and cellular models may be important pharmacogenomic methods to rapidly characterize variants of potential relevance in additional drug-gene pathways.

Citation Format: Shikshya Shrestha, Steve Offer, Robert B. Diasio. Functional analysis of rare dihydropyrimidine dehydrogenase variants relevant to 5-fluorouracil toxicity. [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 5487. doi:10.1158/1538-7445.AM2015-5487
Abstract 5496: Effect of BTK inhibitors on differentiation of human monocyte-derived dendritic cells
Authors: Sun Zhijian , Zhou Dongping , Luo Lusong
Published: August 2015

Dendritic cells (DCs) are primary antigen-presenting cells (APCs), which process tumor antigen and present it on the cell surface to the T cells of the adaptive immune system. However, immature DCs facilitate tolerance toward cancer cells. Bruton's tyrosine kinase (BTK) is highly expressed in B cells, monocytes, macrophages and dendritic cells and plays crucial roles in differentiation and activation of myeloid cells. Dendritic cells in Btk−/− mice were reported to have more mature phenotype and stronger in vitro and in vivo T cell stimulatory ability than wild-type DCs. However, the functions of BTK in human DC differencation and maturation were not well characterized. In this study, we revealed the effect of BTK inhibition with ibrutinib, an FDA approved drug for chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), in human monocyte derived dendritic cells (moDCs). We show that BTK was expressed in monocytes and moDCs. BTK activity was regulated during moDCs differencation and BTK inhibitors blocked GM-CSF/IL4 induced activation of BTK. LPS instead of CD40L treatment increased BTK phosphorylation. It was noted that the percentage of CD11c+ MHC IIlow subsets, which represented the immature DCs, was significantly reduced upon ibrutinib treatment during moDC differencation. Meanwhile, ibrutinib dose-dependently increased proportion of CD11c+ MHC IIhigh cells, which represent more mature DCs. We also tested BGB-3111, a more selective BTK inhibitor without ITK, TXK, EGFR, HER2 and JAK3 activity in the same assay. We observed increased MHC II level during differentiation of human moDCs with BGB-3111 treatment. These results support combination strategies for BTK inhibitors with other cancer immunotherapy agents.

Citation Format: Zhijian Sun, Dongping Zhou, Lusong Luo. Effect of BTK inhibitors on differentiation of human monocyte-derived dendritic 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 5496. doi:10.1158/1538-7445.AM2015-5496
Abstract 55: Over-expression of FoxM1 in breast cancer can be therapeutically targeted using thiostrepton
Authors: Ahmed Maqbool , Hussain Azhar , Begum Rafiya , Thangavel Saravanan , Ajarim Dahish S. , Beg Shaham , Uddin Shahab , Al-Kuraya Khawla S.
Published: August 2015

Breast cancer incidence is increasing worldwide and is one of the most common cancers among women in Saudi Arabia. In spite of conventional treatment regimes in breast cancer, the prognosis is still poor. However, over the last decade, outcome of breast cancer has improved due to increased awareness among the population leading to early detection. Forkhead box protein 1 (FoxM1), is an important member of Forkhead family protein, plays an important role in the pathogenesis of various cancer including basal cell carcinoma. In addition, FoxM1 has also been shown to be involved in the pathogenesis of breast cancer from other regions. However, the role of FoxM1 in Saudi Arabia breast cancer has not been fully elucidated. Therefore in this study, we first evaluated the expression of FoxM1 in a panel of patients with breast cancer samples in Saudi Arabia by immuno histo chemistry. Our data showed that FoxM1 is over expressed in 79% (770/975) of breast cancer and directly associated with clinical parameters such as younger age (<30yrs)(p = 0.00172),high Grade(p<0.0001), mucinous histology (p<0.0001), and triple negative phenotype (p<0.0001). In addition, FoxM1over-expession was also significantly associated with activated AKT (p = 0.0001), as well as high Ki67 expression(p<0.0001),matrix metalloproteinase-9 (MMP-9) (p <0.0001), and XIAP (p <0.0001). In vitro, using breast cancer cell lines, we found that treatment with thiostrepton resulted in inhibition of cell viability and induction of apoptosis in a dose dependent manner. In addition, in these cell lines, thiostrepton treatment caused down regulation of FoxM1, in activation of AKT and decreased the expression of XIAP, and MMP-9. Furthermore pharmacological inhibition of FoxM1 triggered Caspase dependent apoptosis in breast cancer cell lines. Altogether, our results suggest that FoxM1 and its associated signaling pathway may be a potential target for therapeutic intervention in the treatment of breast cancer.

Citation Format: Maqbool Ahmed, Azhar Hussain, Rafiya Begum, Saravanan Thangavel, Dahish S. Ajarim, Shaham Beg, Shahab Uddin, Khawla S. Al-Kuraya. Over-expression of FoxM1 in breast cancer can be therapeutically targeted using thiostrepton. [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 55. doi:10.1158/1538-7445.AM2015-55
Abstract 550: Evaluation of NQO1 as a potential diagnostic marker for bladder cancer
Authors: Cuff Simone , Lewis Ruth , Jaffar Mohammed , Knox Richard , Weeks Ian
Published: August 2015

The present method for detection of bladder cancer involves ultrasonography followed by cystoscopy. This process is time intensive, requires skilled personnel and is unpleasant for the patient as a result of its invasive nature. This results in patient drop-out and patients that have recovered from bladder cancer are lost to follow-up due to the necessity for hospital visits for monitoring. We have used a novel test in order to monitor NAD(P)H quinone oxidoreductase 1 (NQO1) activity in cells recovered from routine urine samples since expression of NQO1 has previously been reported to be increased in several tumour types including transitional cell carcinoma of the bladder.

Informed consent and urine samples were obtained from 73 bladder cancer patients and 42 age-matched bladder cancer-negative outpatients at the University Hospital of Wales, Cardiff. Live exfoliated cells were collected from the urine and assessed for their ability to metabolise an NQO1-specific substrate into a highly fluorescent product. Fluorescence intensity was then measured in a simple, inexpensive instrument. Results were checked against clinical correlates, bladder cancer stage, age, medication, and smoking status. Initial tests in cell lines showed excellent correlation with NQO1 levels detected by other methods. We found that in patient samples, while cell numbers in urine and expression of NQO1 per cell were not in themselves significant, NQO1 per ml of urine was highly significantly increased in bladder cancer patients (p = 0.009). Further, NQO1 was higher in patients that had high grade tumours as described in the WHO 2004 guidelines (p = 0.003).

The sensitive fluorescence-based method to detect NQO1 activity in exfoliated urothelial cells shows exceptional promise in the development of rapid, non-invasive bladder cancer diagnostics. The method will be used on larger numbers of samples in order to be able to verify these initial findings.

Note: This abstract was not presented at the meeting.

Citation Format: Simone Cuff, Ruth Lewis, Mohammed Jaffar, Richard Knox, Ian Weeks. Evaluation of NQO1 as a potential diagnostic marker for bladder 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 550. doi:10.1158/1538-7445.AM2015-550
Abstract 5506: Anti-angiogenic effects of zoledronic acid on osteotropic breast cancer cells
Authors: Jackson Cynthia , Whalen William , Morshedian Shana , Bush Jason A.
Published: August 2015

Emerging data suggest the bisphosphonate, zoledronic acid (ZOL), exerts both indirect and direct anti-tumor effects by decreasing tumor cell proliferation, increasing apoptosis, and inhibiting angiogenesis. Recent data indicate that antiangiogenic factors exert cellular effects via suppression of mitochondrial oxidative phosphorylation/respiration and elevation of intracellular ROS levels. This project intended to define proteins involved in response to ZOL-treatment of an osteotropic breast cancer cell line, MDA-231BO. Results demonstrate that MDA-231BO cells are more sensitive to ZOL cytotoxicity and salutary effects are both media- and calcium-dependent. This cytotoxicity was more pronounced in spheroid cultures. To investigate pro-apoptotic mechanisms of ZOL, total RNA, whole cell protein lysates, or mitochondrial-enriched fractions were isolated from MDA-231BO cells and using a combination of RT-PCR, 1D/2D-PAGE separation and MALDI-MS/MS, we identified several angiogenesis inhibitors that were upregulated and pro-angiogenic factors that were downregulated after ZOL treatment. Functional studies demonstrate the improved efficacy of ZOL on MDA-231BO compared to MDA-MB-231 controls. However, when the cells were challenged in a hypoxic environment, these effects were mitigated. Taken together, these results further support a model that ZOL tips the ‘angiogenic switch’ in favor of anti-angiogenic factors in cancer cells with a propensity for bone metastases.

Citation Format: Cynthia Jackson, William Whalen, Shana Morshedian, Jason A. Bush. Anti-angiogenic effects of zoledronic acid on osteotropic breast 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 5506. doi:10.1158/1538-7445.AM2015-5506
Abstract 5518: Development of bio-affinity nanoparticles: Applications for cancer imaging and therapeutics
Authors: Dotan Idit , Roche Philip J.R. , Mitmaker Elliot , Trifiro Mark A. , Paliouras Miltiadis
Published: August 2015

Treatment of advanced cancer, is an extremely challenging proposition. Surgery is curative at early stages, but with substantial complications. Molecular treatments in early disease are of questionable benefit, coupled with the lack of therapeutic options in advanced disease, make treatment extremely challenging. Early cancers evolve through many somatic mutations involving many selective processes, induced in part by classical drug therapies themselves initiating drug resistance. Genetic cancers studies show extensive single missense mutations, copy number variations, splicing variants, genetic rearrangements and short DNA alterations in a large number of genes. However, physical agent therapies are immune to genetic alterations, a targeting the cancer cell and adjacent cells. By investigating cell surface markers that are correlated with disease progression, we have coupled bio-affinity agents (antibody, ligand, aptamer) to functionalized carbon nanoparticles and developed a platform of delivering highly-localized “energy” in the immediate proximity to the cells causing significant cellular damage. Similarly, these agents can be simultaneously used as imaging agents, creates a theranostic treatment agent. We have used papillary thyroid (PTC) and prostate cancer (CaP) cancer cells and selectively targeted THSR and PSMA, respectively in our models. Using a 2W laser light for 30 seconds, we are able to achieve near 100% cell killing of targeted cells, whereas receptor-null cells remain unharmed. Electron microscopy was used as a direct analysis to answer the mechanistic question of cell necrosis and cell death that cannot be approached via light microscopy. Altogether these results suggest that significant and specific cell killing can be achieved with small amounts of power over short periods of time. In conclusion, whereas in the past, simultaneous multiple drugs therapies have been limited by systemic toxicities, this approach represents a single delivery platform that can carry several small molecule drugs, in conjunction with new genetic modalities, in a targeted fashion with the added benefit of self-imaging so as to document successful targeting.

Citation Format: Idit Dotan, Philip J.R. Roche, Elliot Mitmaker, Mark A. Trifiro, Miltiadis Paliouras. Development of bio-affinity nanoparticles: Applications for cancer imaging and therapeutics. [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 5518. doi:10.1158/1538-7445.AM2015-5518
Abstract 5519: Engineered protein nanocage for targeted delivery of siRNA to cancer cells
Authors: Lee Eun Jung , Yang Yoosoo , Kim In-san , Kim Kwangmeyung , Lee Jeewon
Published: August 2015

Considering the problems of small interfering RNA (siRNA) delivery using traditional viral and non-viral vehicles, a new siRNA delivery system to enhance efficiency and safety needs to be developed. Here we genetically engineered human ferritin based protein nanocage to simultaneously display various functional peptides on the surface of protein nanocage: cationic peptide to capture siRNA, tumor cell targeting and penetrating peptides, and enzymatically cleaved peptide to release siRNA inside tumor cell. In particular, we easily changed the tumor cell targeting peptide depending on target moiety [epidermal growth factor receptor or vimentin in this case] on the tumor cell surface. The polymerized siRNA (poly siRNA) tightly bound to the engineered protein nanocage and formed stable and condensed structure (poly siRNA-protein nanocage complex) without cytotoxicity problem. Furthermore, siRNAs in the condensed complex were effectively protected from endonuclease due to a shielding effect of protein nanocage. In the in vitro treatment of poly siRNA-protein nanocage complex, both of the tumor cell targeting and penetrating peptides were important for efficient delivery of siRNA, and the red fluorescent protein (RFP) expression in RFP-expressing tumor cells was notably suppressed by the delivered siRNA with the complementary sequence to RFP mRNA. It seems that the human ferritin-based protein nanocage is an efficient, stable, and safe tool for siRNA delivery, having a great potential for application to in vivo cancer treatment. The unique feature of protein nanocage is that multiple and functional peptides can be simultaneously and evenly placed and also easily switched on the protein nanocage surface through a simple genetic modification, which is likely to make protein nanocage appropriate for targeted delivery of siRNA to a wide range of cancer cells.

Citation Format: Eun Jung Lee, Yoosoo Yang, In-san Kim, Kwangmeyung Kim, Jeewon Lee. Engineered protein nanocage for targeted delivery of siRNA to 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 5519. doi:10.1158/1538-7445.AM2015-5519
Abstract 5523: A novel aptamer targeting agent for prostate cancer
Authors: Powell Gray Bethany , Kelly Linsley , Levy Matthew , Sullenger Bruce A.
Published: August 2015

Background: Prostate cancer is both the most common cancer diagnosis and the second most common cause of cancer-related death for men in the United States. An ideal new therapy would selectively deliver the drug to the tumor while also preventing toxicity in normal tissue.

Methods and Results: Recent exciting clinical advances with antibody drug conjugates have resulted in FDA approval of such tumor-targeting therapies. However, antibodies are difficult to chemically modify, require humanization, and their large size hinders tumor penetration. Aptamers - small nucleic acid ligands - have many advantages over traditional antibody targeting agents, including ease of synthesis, amenability to chemical modification, and reversibility. Moreover, they exhibit antibody-like target affinities and specificities at a fraction of the size, allowing more efficient tumor penetration.

We have identified an aptamer, termed E3, which selectively internalizes into prostate cancer cells, but does not target normal cells. Additionally, E3 targets and internalizes into a variety of other cancer types, including breast, ovarian, lung, colon, melanoma, glioblastoma, liver, and leukemia cancers. Linking the E3 aptamer to a deimmunized version of the Pseudomonas exotoxin (PE38) kills prostate cancer cells in vitro with an IC50 of 0.4-1.1 nM while displaying no measured toxicity towards normal cells, and linking E3 to the highly toxic auristatin drugs, MMAE or MMAF, kills prostate cancer cells in vitro with an IC50 of 5-12 nM. Significantly, E3 maintains its tumor-targeting abilities in vivo, targeting both prostate tumors and patient-derived colon cancer tumors in mice as evidenced by near-infrared in vivo imaging.

Conclusions: Together, these studies show the potential therapeutic benefit of E3 drug targeting for the treatment of prostate cancer.

Citation Format: Bethany Powell Gray, Linsley Kelly, Matthew Levy, Bruce A. Sullenger. A novel aptamer targeting agent for prostate 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 5523. doi:10.1158/1538-7445.AM2015-5523
Abstract 5525: Antibody/drug-conjugated micelle as a versatile platform technology for targeted tumor delivery
Authors: Harada Mitsunori
Published: August 2015

Background:

Antibody-drug conjugates (ADCs) have been recognized as a promising anticancer agent, despite their stringent requirements of antibody affinity, internalization, payload potency, and linker stability. To drive the next generation of ADCs and improve versatility of the component combination, we have designed, prepared and recently developed Antibody/Drug-Conjugated Micelle (ADCM) system. ADCM is composed of polyethylene glycol-poly (amino acid derivative) polymers, which can form a micellar nanoparticle spontaneously in aqueous media with a diameter of 20-100 nm. Antibodies are attached to the surface of the nanoparticle, while payloads are incorporated in the inner core at a payload-to-antibody molecular ratio of 100-200. In this study, monoclonal antibody, trastuzumab or cetuximab, and anticancer drug, anthracycline antibiotic epirubicin or novel hemiasterlin analogue E7974, were used as a targeting sensor and a payload of the ADCM, respectively. Here we report the results of in vitro and in vivo studies.

Methods:

The ADCMs were prepared as described previously (Japan Patent No.4538666) with slight modification. To examine the cell binding behavior, flow cytometry analysis was performed using an Accuri C6 system. The antigen affinity was evaluated using a Biacore system. The cellular uptake was examined fluorescently using ADCMs employing Alexa 488-labeled antibodies. To compare the in vivo antitumor effects, ADCM, untargeted micelle and the payload were administered intravenously 3 times weekly at the same payload-equivalent dose to nude mice bearing human tumor xenografts with HER2 (NCI-N87 gastric or trastuzumab-resistant JIMT-1 breast tumor) or EGFR (BxPC-3 pancreatic or LS174T colon tumor) expression. Tumor volumes and animal body weights were monitored 2 or 3 times a week.

Results:

The ADCMs showed similar cell-binding behavior and antigen affinity to the original antibodies. They were found to be internalized more efficiently than the original antibodies. In vivo efficacy studies demonstrated that the ADCMs inhibited tumor growth most effectively compared to control formulations. The therapeutic indexes were significantly improved in terms of the maximum tolerated dose/ED50 (50% effective dose) compared to untargeted micelles and native payloads. These results suggest the potential clinical application of the ADCMs.

Conclusion:

These data showed improved versatility of the component combination and increased therapeutic indexes of ADCM system, indicating a promising next generation of ADCs.

Citation Format: Mitsunori Harada. Antibody/drug-conjugated micelle as a versatile platform technology for targeted tumor delivery. [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 5525. doi:10.1158/1538-7445.AM2015-5525