bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 307: Targeting BCL-2 and BCL-xL with a novel dual inhibitor APG-1252 triggers cell death and inhibits tumor growth in small cell lung cancer models
Authors: Wang GuangFeng , Min Ping , Wu MiaoYi , Dang Shuo , Tang ChuanYan , Zhang Fei , Guo Ming , Wang Shaomeng , Deng Jing , Fang Douglas D. , Yang DaJun , Zhai YiFan
Published: August 2018

Small cell lung cancer (SCLC) is one of the most deadly diseases with a dismal five year survival rate less than 7%. Even though most SCLC patients respond to the initial platinum-based cytotoxic or radiation therapies, they inevitably relapse and succumb to the disease. Anti-apoptotic proteins BCL-2 and BCL-xL, which are highly expressed in 40-60% of SCLCs and play a critical role in tumorigenesis and drug resistance, have been emerging as a promising target for therapeutic intervention. We have recently developed a novel dual BCL-2/BCL-xL inhibitor APG-1252 for cancer therapy. In this study, the effect of APG-1252 was evaluated in a panel of SCLC cell lines for discovery of indications and predictive biomarkers. The results show that the sensitivity of SCLC cell lines with sub-µM or nM IC50 values are correlated with the higher expression levels of BCL-2/BCL-xL, BIM and/or PUMA but lower levels of MCL-1. Conversely, the resistant cell lines either lack of BCL-2/BCL-xL protein, or exhibit higher level of MCL-1 protein. In xenograft tumor models, consistent with in vitro results, APG-1252 exhibits antitumor activities in the models derived from the sensitive cells but not in those from the resistant cells. Interestingly, while ABT-263 failed to inhibit H146 xenograft tumor growth, despite of its similar in vitro killing ability as APG-1252, APG-1252 showed potent antitumor activity in the xenograft model. To overcome the drug resistance conferred by MCL-1 in the resistant cells, we explore the combination therapy with other targeted agents. We found that our novel MDM2 inhibitor APG-115 was able to overcome the intrinsic resistance and sensitize those cells to APG-1252 in vitro, suggesting that reducing the apoptotic threshold by inhibiting other anti-death proteins like MCL-1 or increasing apoptotic function through p53 can enhance SCLC sensitivity to APG-1252. Collectively, APG-1252 represents a novel opportunity that can neutralize the protection from BCL-2/BCL-xL and trigger cell death and inhibit tumor growth in SCLC models. With the significance of these preclinical data, APG-1252 has been granted for phase 1 clinical trials in USA (NCT03080311) and China.

Citation Format: GuangFeng Wang, Ping Min, MiaoYi Wu, Shuo Dang, ChuanYan Tang, Fei Zhang, Ming Guo, Shaomeng Wang, Jing Deng, Douglas D. Fang, DaJun Yang, YiFan Zhai. Targeting BCL-2 and BCL-xL with a novel dual inhibitor APG-1252 triggers cell death and inhibits tumor growth in small cell lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 307.
Abstract 3072: MiRNA-2861 behaves as LCSC biomarker and regulates the HDAC5-ERK signal pathway
Authors: Zhu Yimin , Zhao Mengya , Li Lin
Published: August 2018

Cancer stem cells (CSCs) are responsible for cancer initiating, recurrence and drug-resistance. Discovery of novel biomarkers for CSCs is helpful for early diagnosis and prognosis. In our study, the important role of miR-2861 in maintaining the stemness of LCSCs was investigated. The LCSCs differentiation model was established through introducing serum into the medium of H460 spheres. MiR-2861 expression was significant higher in LCSCs no matter compared to the differentiation cells or normal cells. HDAC5 expression was positively correlated with miR-2861 in LCSCs and knockdown of miR-2861 decreased the expression of HDAC5, which implied that HDAC5 may be involved in the differentiation of LCSCs mediated by miR-2861.The role of HDAC5 in the regulation of LCSCs differentiation was further verified by the inhibitory effect of LMK-235 on the phosphorylation of ERK1/2, which was recognized as the regulator of CSCs differentiation. Our study provided a better understanding of miR-2861 and HDAC5 axis in maintaining the stemness of LCSCs and laid a foundation for molecular targeted therapy.

Citation Format: Yimin Zhu, Mengya Zhao, Lin Li. MiRNA-2861 behaves as LCSC biomarker and regulates the HDAC5-ERK signal pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3072.
Abstract 3079: Epstein Barr virus-encoded LMP1 reprograms glucose metabolism to enhance cell motility in nasopharyngeal epithelial cell
Authors: Zhang Jun , Lin Weitao , Tsang Chi Man , Liu Teng Fei , Tang Wing Chong , Yip Yim Ling , Deng Wen , Jia Lin , Tsao Sai Wah
Published: August 2018

How oncogenic virus rewires glucose flux to regulate cell motility is not well-understood. Epstein-Barr virus (EBV) infection of preinvasive nasopharyngeal epithelium is believed to be an essential initiation event during nasopharyngeal carcinoma pathogenesis. Here we demonstrate that EBV-encoded LMP1 enhances glycolysis thereby accelerating cell mobility of nasopharyngeal epithelial (NPE) cells. Activation of IGF1/mTORC2/AKT/PDHE1α signaling which mediates Histone 3 acetylation is involved in cell motility in EBV-infected NPE cells. Blocking the glucose uptake by pharmacological inhibitors effectively suppress glycolysis as well as the cell motility, linking the glucose metabolism to cell movement. We further show that mTORC2/AKT mediated phosphorylation of pyruvate decarboxylase kinase 1 (PDHK1) and its substrate, pyruvate decarboxylase E1α (PDHE1α) component plays a significant role in cell motility. PDHE1α knockdown effectively blocks LMP1-induced cell motility while overexpression of its phosphomimetic mutant S293A but not S293D inhibits LMP1-induced cell motility, supporting the involvement of PDHE1α in enhanced cell motility is depended on Ser293 phosphorylation. Furthermore, PDHE1α translocation into nucleus enhances Histone 3 acetylation and its binding to the promoter region of snail to enhance cell motility. Finally, we find that LMP1 increases the mRNA expression and the secretion of the ligand IGF1, which promotes phosphorylation of IGF1R as the key regulator for LMP1-induced mTORC2/AKT activation. In summary, this study provides evidences of involvement of EBV infection in metabolic reprogramming of NPE cells to promote cell motility involving activation of mTORC2/AKT/PDHE1α.

Acknowledgment: This project was supported by funding from the Research Grants Council, Hong Kong: General Research Fund (17120814, 17161116, 17111516, 171110315), Area of Excellence (AoE/M-06/08), Theme-Based Research Scheme (T12-401/13-R), Collaborative Research Fund (Project reference: C7027-16G); Health and Medical Research Fund of Hong Kong (12110782, 13120872, 04151726); and CRCG, University of Hong Kong, Hong Kong.

Citation Format: Jun Zhang, Weitao Lin, Chi Man Tsang, Teng Fei Liu, Wing Chong Tang, Yim Ling Yip, Wen Deng, Lin Jia, Sai Wah Tsao. Epstein Barr virus-encoded LMP1 reprograms glucose metabolism to enhance cell motility in nasopharyngeal epithelial cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3079.
Abstract 3084: Epstein Barr virus-encoded LMP1 activates the mTORC2 signaling pathway to reprogram glucose metabolism in nasopharyngeal epithelial cell
Authors: Zhang Jun , Lin Weitao , Tsang Chi-Man , Liu Teng Fei , Tang W C. , Yip Tim Ling , Deng Wen , Jia Lin , Tsao Sai-Wah
Published: August 2018

Epstein-Barr virus (EBV) infection of preinvasive nasopharyngeal epithelium is believed to be an essential initiation event during nasopharyngeal carcinoma pathogenesis, but the underlying mechanisms are largely undefined. Here we demonstrate that EBV-encoded LMP1 reprograms the glucose metabolism from oxidative phosphorylation to glycolysis of nasopharyngeal epithelial (NPE) is a major process for NPC pathogenesis. RNA-seq analysis indicates that EBV infection reprograms the glucose metabolism from glycolysis and oxidative phosphorylation and enhances the metabolic associated pathways. Our study show EBV encoded LMP1 is important for this event, EBV infection and LMP1 expression enhance the extracellular acidification rate and decrease the oxygen consumption rate in NPE cells. We further show that activation of mTORC2/AKT signaling is involved in this metabolic reprogramming in EBV-infected NPE cells. Genetic or pharmacologic inhibition of the mTORC2/AKT signaling blocks the LMP1-induced glycolysis. In summary, this study provides evidences of involvement of EBV infection in metabolic reprogramming of NPE cells. Acknowledgment: This project was supported by funding from the Research Grants Council, Hong Kong: General Research Fund (17120814, 17161116, 17111516, 171110315), Area of Excellence (AoE/M-06/08), Theme-Based Research Scheme (T12-401/13-R), Collaborative Research Fund (Project reference: C7027-16G); Health and Medical Research Fund of Hong Kong (12110782, 13120872, 04151726); and CRCG, University of Hong Kong, Hong Kong.

Citation Format: Jun Zhang, Weitao Lin, Chi-Man Tsang, Teng Fei Liu, W C. Tang, Tim Ling Yip, Wen Deng, Lin Jia, Sai-Wah Tsao. Epstein Barr virus-encoded LMP1 activates the mTORC2 signaling pathway to reprogram glucose metabolism in nasopharyngeal epithelial cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3084.
Abstract 3086: CPAP enhances diethylnitrosamine-induced hepatocarcinogenesis via STAT3 pathway
Authors: Hung Liang-Yi
Published: August 2018

Chronic and persistent inflammation is a well-known carcinogenesis promoter. Hepatocellular carcinoma (HCC) is one of the most common inflammation-associated cancers; most HCC arises in the context of chronic inflammation and hepatic injury. Both NF-κB and STAT3 are important regulators for inflammation. Our previous studies indicated that CPAP (centrosomal P4.1-associated protein), a centrosomal protein that majorly participates in centrosome functions, is overexpressed in HCC and can increase TNFα-mediated NF-κB activation and IL-6-induced STAT3 activation. A hepatocyte-specific CPAP-expressing transgenic mouse was generated to investigate the physiological role of CPAP in hepatocarcinogenesis. An obvious inflammatory cell accumulation and fatty change were shown in the CPAP transgenic (Tg) mouse liver. The ALT level as well as the expression of inflammatory genes, such as IL-6, IL1β and TNF-α in CPAP Tg mouse are higher than in the wild type (WT) mouse. By high-dose and short-term diethylnitrosamine (DEN) treatment, the ALT level, pro-inflammatory genes and STAT3 activation are increased in CPAP Tg mouse; low-dose and long-term DEN treatment induces more severe liver tumor formation in CPAP Tg mouse than in WT mouse. In summary, these results suggest that CPAP may promote hepatocarcinogenesis through enhancing the inflammation pathway via activating the STAT3 pathway.

Citation Format: Liang-Yi Hung. CPAP enhances diethylnitrosamine-induced hepatocarcinogenesis via STAT3 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3086.
Abstract 3090: Phenol modifying influence on genotoxic effect in organism during peroral combined administration with benzo[a]pyrene
Authors: Ostash Olga , Chernichenko Igor , Balenko Nina , Vinarska Elena , Grigorenko Lyudmila , Lukyanchuk Svetlana , Shvager Oksana
Published: August 2018

In the modern world in different spheres of life, people experience a load of the complex of numerous chemical compounds of different types, which have a wide range of biological effects - from toxic ones to mutagenic and carcinogenic. For instance, people suffer from the growth of cancer, which is one of the direct consequences of the impact of environmental pollution by chemical compounds. It has now been proved that the development of cancer is determined not only by the initiating effect of chemical carcinogens, but also by the modifying effect of the concomitant toxic compounds. The aim of this work was experimental study of the role of toxic substances (phenol) in the modification of the evidence of genotoxic and immunological changes in organism in the early stages of carcinogenic activity (benzo[a]pyrene).The studies were conducted with random-bred white male mice. Peroral administration was applied to animals taken benzo[a]pyrene (day-dose 0.1mg), benzo[a]pyrene - phenol (day-dose 0.1; 0.002 mg). Genotoxic changes in forestomach were assessed with the help of micronuclear test. The carcinogenic effect (forestomach papillomas) and common regularities of genotoxical and immunological changes in relation to carcinogenesis and their dose and treatment duration dependence under benzo[a]pyrene (day-dose 0,1mg), benzo[a]pyrene and phenol (day-dose 0,1; 0,002 mg) complexes oral administration in random-bred white mice were established. The noted regularities conducted in interrelation and identical directivity of mutagenic (increasing of micronucleus incidence) and immunological (reducing the relative number of T-lymphocytes) indexes changes in relation to carcinogenesis, presence of reliable cross-correlation connections between them in the end of the first months. Besides, the modifying effect of phenol on the carcinogenesis that appeared by increasing of micronucleus incidence, immunosuppression and index of forestomach papillomas multiplicity was determined. The findings detect the presence of cancer risk from the action of carcinogenic and toxic substances, the formation of which begins in the early period (at the end of the first month) and manifests itself as a genotoxic effect in combination with immunosuppression, which must be taken into account in the hygienic assessment of the state of the environment.

Citation Format: Olga Ostash, Igor Chernichenko, Nina Balenko, Elena Vinarska, Lyudmila Grigorenko, Svetlana Lukyanchuk, Oksana Shvager. Phenol modifying influence on genotoxic effect in organism during peroral combined administration with benzo[a]pyrene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3090.
Abstract 3110: Heterokaryon formation and nuclear fusion of hybridizing cancer cells visualized in real time by color-coded imaging
Authors: Suetsugu Atsushi , Matsumoto Takuro , Hasegawa Kousuke , Nakamura Miki , Kunisada Takahiro , Shimizu Masahito , Saji Shigetoyo , Moriwaki Hisataka , Bouvet Michael , Hoffman Robert M.
Published: August 2018

Background: Fusion of cancer cells has been studied for over half a century. However, the steps involved after initial fusion between cells, such as heterokaryon formation and nuclear fusion, have been difficult to observe in real time. In order to be able to visualize these steps, we have established cancer-cell sublines from the human HT-1080 fibrosarcoma, one expressing green fluorescent protein (GFP) linked to histone H2B in the nucleus and a red fluorescent protein (RFP) in the cytoplasm and the other subline expressing RFP in the nucleus (mCherry) linked to histone H2B and GFP in the cytoplasm.

Materials and Methods: The two reciprocal color-coded sublines of HT-1080 cells were fused using the Sendai virus. The fused cells were cultured on plastic and observed using an Olympus FV1000 confocal microscope.

Results: Multi-nucleate (heterokaryotic) cancer cells, in addition to hybrid cancer cells with a single fused nucleus undergoing mitosis, were observed by confocal microcopy. Red, green, orange and yellow nuclei were observed in cells. The orange and yellow nuclei indicate nuclear fusion. Red and green nuclei remained unfused.

Conclusion: Cell fusion with subsequent heterokaryon formation followed by nuclear hybridization may be an important natural phenomenon between cancer cells that may make them more malignant. The ability to image the complex processes following cell fusion using reciprocal color-coded cancer cells will allow greater understanding of the genetic basis of malignancy.

Citation Format: Atsushi Suetsugu, Takuro Matsumoto, Kousuke Hasegawa, Miki Nakamura, Takahiro Kunisada, Masahito Shimizu, Shigetoyo Saji, Hisataka Moriwaki, Michael Bouvet, Robert M. Hoffman. Heterokaryon formation and nuclear fusion of hybridizing cancer cells visualized in real time by color-coded imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3110.
Abstract 3111: Expansion of mouse prostate epithelial stem cells in serum-free ProstaCult Organoid Growth Medium
Authors: Stingl John , Rowbotham David , Thomas Terry E. , Eaves Allen C. , Louis Sharon A.
Published: August 2018

Growing prostate epithelial cells as organoids in a three-dimensional (3D) cell culture environment represents a more physiological model system than conventional 2D adherent cell culture systems for studying many different aspects of prostate epithelial cell biology. Two types of prostate epithelial organoids have been previously described: large hollow cystic organoids, and smaller solid organoids (Karthaus et al, Cell 2014). Both types of organoids are composed of androgen receptor (AR)-expressing luminal cells and keratin (K) 5-expressing basal cells, but only vary in the proportion of each cell type, with the cystic organoids enriched for luminal cells and solid organoids enriched for basal cells. We are currently developing ProstaCult™ Organoid Growth Medium, a serum-free medium for the long-term propagation of both types of organoids from mouse prostate tissue. To initiate the cultures, mouse prostates are enzymatically dissociated sequentially in collagenase type II, trypsin and dispase to generate a single cell suspension, and 5x103 the liberated cells are then embedded in Corning® Matrigel® and cultured in ProstaCult™ Organoid Growth Medium. Approximately 10±2% (mean±sem; n=6) of the seeded cells will proliferate and generate approximately equivalent numbers of cystic and solid organoids, which can be dissociated and passaged every 7 days as a single cell suspension. Typical total epithelial cell expansion observed during media development ranges from 21-240-fold per passage for a minimum of 4 passages. Experiments are ongoing to determine the long-term passageability of organoids maintained in ProstaCult™ Organoid Growth Medium. Organoids that are generated at multiple passages are composed of a polarized epithelium with AR+K18+ luminal cells and K5+ basal cells. These results demonstrate that ProstaCult™ Organoid Growth Medium efficiently generates and expands prostate epithelial organoids.

Citation Format: John Stingl, David Rowbotham, Terry E. Thomas, Allen C. Eaves, Sharon A. Louis. Expansion of mouse prostate epithelial stem cells in serum-free ProstaCult Organoid Growth Medium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3111.
Abstract 3112: Distinct oncogenic events induce different DNA methylation and copy number changes in human mammary epithelial cells
Authors: manoir Stan P. du , Fonti Claire , Saumet Anne , Abi-Khalil Amanda , Orsetti Béatrice , Elouan Cleroux , Bender Ambre , Dumas Michael , Colinge Jacques , Weber Michael , Theillet Charles
Published: August 2018

Gene expression differences, combined with distinct patterns of genomic rearrangements and epigenetic modifications constitute the bases of molecular classification of breast cancer. Molecular subtypes may originate from different cell lineages in the mammary gland, but also from the early activation of oncogenes that may drive the establishment of these molecular subtypes. However, in the natural history of human cancer, it is difficult to discriminate between these two factors : cell lineage and initial oncogenic alterations. In this work, we designed an experimental strategy aiming at determining whether activation of distinct oncogenic pathways in human mammary epithelial cells (HMEC) could lead to different patterns of genetic and epigenetic changes. We show that initial activation of CCNE1, WNT1 and RASv12 in shp53 immortalized HMECs results in different and reproducible profiles of mRNA and miR expression, copy number alterations (CNA) and DNA methylation modifications. Interestingly, first the extend of CNA measured as fraction of the genome altered was lower in HMECs transformed by RAS than CCNE1 and WNT1 transformed HMECs revealing a lower genetic instability. This was confirmed by less numerous γH2Ax and 53BP1 nuclear foci in HMECs transformed by RAS. Second, HMECs transformed by RAS bore specific profiles of CNAs and DNA methylation, clearly distinct of those shown by CCNE1 and WNT1 transformed HMECs. Genes differentially expressed in the RAS and the CCNE1/WNT1 clusters and included in CNAs or with variable CpG methylation were mostly different, depicting the activation of distinct signaling pathways in these two clusters. These data indicate that early activation of distinct oncogenic pathways may leads to adaptive events resulting in specific pattern of CNAs and DNA methylation changes. We postulated that the early activation of oncogenes may be an important determinant of the establishment of breast cancer molecular subtypes along with cell lineage origin.

Citation Format: Stan P. du manoir, Claire Fonti, Anne Saumet, Amanda Abi-Khalil, Béatrice Orsetti, Cleroux Elouan, Ambre Bender, Michael Dumas, Jacques Colinge, Michael Weber, Charles Theillet. Distinct oncogenic events induce different DNA methylation and copy number changes in human mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3112.
Abstract 3114: In vitro culture system for circulating tumor cell
Authors: Yin-Ju Chen , Lu Long-Sheng , Lee Hsin-Lun , Ting Lai-Lei , Chiou Jeng-Fong
Published: August 2018

Circulating tumor cells (CTCs) detection could be considered a real-time “liquid biopsy” approach and contains several advantages such as being minimally invasive, easy and safe to perform, multiple samples can be taken over time, better prognosis to indicate an elevated risk of metastases, improved therapy monitoring, and providing live disease status information. CTC analysis is a promising new diagnostic field for estimating the risk for metastatic relapse and metastatic progression in patients with cancer. However, the number of CTCs is very low so the establishment of cell culture from CTCs becomes the most challenging over the past year. In this study, we development a cost-effective and reproducible in vitro system for primary CTC cultures or established cell lines from CTCs. 8 CTC cell lines were generated from patients including breast cancer, small cell lung cancer, pancreatic cancer and expanded for more than one year. These cell lines exhibit the tissue-specific markers and also correlated with clinical treatment response from patients. This system provides an opportunity for broad applications of CTCs for personalized oncology and advancing precision medicine.

Citation Format: Chen Yin-Ju, Long-Sheng Lu, Hsin-Lun Lee, Lai-Lei Ting, Jeng-Fong Chiou. In vitro culture system for circulating tumor cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3114.
Abstract 3117: Full spectrum of in vitro disease models with optimized CRISPR-Cas9 gene editing in difficult-to-transfect blood-lineage cells
Authors: Jin Huanyu , Yu Zhongsheng , Pak Vladimir , Zhang Yin , Tummala Padmaja , Rajeswaran Pavithra , Nguyen Diana , Maleszewska Monika , Li Jinling , Cao Charles , Chen-Tsai Ruby Yanru
Published: August 2018

CRISPR/Cas9 technology is a powerful tool for gene editing in mammalian cells. However, the efficiency of gene editing in certain types of cells, especially suspension cells such as several blood-lineage cell lines, is extremely low. This is due to a variety of factors including low transfection efficiency of CRISPR/Cas9 reagents, cytotoxicity, and low or undesired gRNA and Cas9 activities in the cell lines. By optimizing the protocol and transfection conditions, we were able to successfully generate a spectrum of gene modifications in blood-lineage cells. Here, we describe techniques optimized for genome editing in some difficult-to-transfect blood-lineage cell lines and the types of modifications achieved: (1) For leukemia cell lines such as KG-1 and MOLM-13 cells, we compared DNA-based versus RNP-based Cas9 delivery efficiency, and found that plasmid-based Cas9 transfection resulted in cytotoxicity. In contrast, the RNP-based route demonstrated significantly lower cytotoxicity and higher efficiency of gene editing. Furthermore, we found that the application of a DNA-dependent protein kinase inhibitor dramatically enhanced the homology directed repair (HDR) in these cell lines; (2) For T lymphocyte cell lines such as Jurkat cells, we could efficiently delete a targeted 5kb regulatory region of a critical gene. We were also able to sequentially knockout (KO) two genes (double KO) in Jurkat cells using a Cas9-plasmid protocol; (3) In the case of the lymphoblastic cell line T2, we successfully created a large fragment knock-in, as well as a gene knockout cell line model, through DNA-based CRISPR/Cas9 delivery. Our studies demonstrate that for CRISPR/Cas9 genome editing in blood-lineage cells, different cell lines may require different approaches or modified protocols to deliver the CRISPR/Cas9 components for efficient and successful modifications of the targeted genes.

Citation Format: Huanyu Jin, Zhongsheng Yu, Vladimir Pak, Yin Zhang, Padmaja Tummala, Pavithra Rajeswaran, Diana Nguyen, Monika Maleszewska, Jinling Li, Charles Cao, Ruby Yanru Chen-Tsai. Full spectrum of in vitro disease models with optimized CRISPR-Cas9 gene editing in difficult-to-transfect blood-lineage cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3117.
Abstract 3128: Immune microenvironment of colorectal tumors and matched liver metastases
Authors: Dagenborg Vegar J. , Marshall Serena Elizabeth , Grzyb Krzysztof , Fretland Åsmund A. , Lund-Iversen Marius , Mælandsmo Gunhild M. , Børresen-Dale Anne-Lise , Ree Anne H. , Edwin Bjørn , Yaqub Sheraz , Flatmark Kjersti
Published: August 2018

Introduction: The presence of tumor-infiltrating lymphocytes, in particular T-cells in the tumor and its microenvironment, has been shown to be associated with prognosis in primary colorectal cancer (CRC) and CRC liver metastases (CLM). In this study we examined T-cell composition in the immune microenvironment in surgical specimens from 64 cases with CRC and resectable CLM.

Methods: Patients were enrolled in the Oslo-CoMet randomized trial (NCT 01516710). CRC and corresponding CLM were examined by immunohistochemistry to quantify all T-cells (Ttot) and subpopulations of cytotoxic (CTL), helper (TH), and regulatory T-cells (Treg). Densities of T-cells and subtypes (cells/mm2) were calculated at T-cell hotspots in the invasive margin (IM) and intra-tumoral (IT) locations. Median densities with interquartile ranges were calculated and compared for each region in CRC and CLM.

Results (Table 1): The highest density of Ttot was observed in the IM for both primary CRC and CLM, with the density in CLM being two-fold higher than CRC (CLMIM median 2853 cells/mm2 vs CRCIM 1257 cells/mm2; p<0.001). For IT regions, the density of Ttot was much lower than in IM, and slightly higher in CRC compared to CLM (CRCIT median 486 vs CLMIT 332; p=0.02). Examining T-cell subtypes, the differences in CTL and TH mirrored Ttot, with the highest densities in the IM (CLMIM>CRCIM) and lower densities in IT regions (CLMIT<CRCIT). For the Treg, the IM densities were similar, while for IT, again, the density was lower in CLM than CRC.

Conclusion: In this study, we analyzed the immune microenvironment in matched CRC and CLM surgical specimens. We are currently analyzing associations with long-term outcome and results will be presented at the conference.

Table 1: Median T-cell density (interquartile range).CRCIMCLMIMCRCITCLMITTtot1257 (931 - 1735)2853 (2305 - 3813)486 (285 - 704)332 (189 - 564)CTL497 (315 - 722)1083 (762 - 1426)106 (51 - 209)99 (49 - 219)nsTH759 (584 - 983)1888 (1449 - 2745)348 (192 - 480)198 (118 - 383)Treg222 (155 - 375)233 (126 - 375)ns144 (75 - 225)43 (22 - 82)CRC and CLM densities at each location (IM and IT) were compared using the Mann-Whitney U test. P-values were <0.05 for all comparisons unless indicated by ns.

Citation Format: Vegar J. Dagenborg, Serena Elizabeth Marshall, Krzysztof Grzyb, Åsmund A. Fretland, Marius Lund-Iversen, Gunhild M. Mælandsmo, Anne-Lise Børresen-Dale, Anne H. Ree, Bjørn Edwin, Sheraz Yaqub, Kjersti Flatmark. Immune microenvironment of colorectal tumors and matched liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3128.
Abstract 313: An Akt3 splice variant lacking the serine 472 phosphorylation site promotes apoptosis and suppresses mammary tumorigenesis
Authors: Hazan Rachel B.
Published: August 2018

The Akt pathway is a well-known promoter of tumor malignancy. Akt3 is expressed as two alternatively spliced variants, one of which lacks the key regulatory serine 472 phosphorylation site. Whereas the function of full-length Akt3 isoform (Akt3/+S472) is well-characterized, that of Akt3/-S472 isoform remains unknown. Despite being expressed at a substantially lower level than Akt3/+S472 in triple negative breast cancer cells, specific ablation of Akt3/-S472, enhanced, whereas overexpression, suppressed mammary tumor growth-consistent with a significant association with patient survival duration relative to Akt3/+S472. These effects were due to striking induction of apoptosis, which was mediated by Bim upregulation, leading to conformational activation of Bax and caspase-3 processing. Bim accumulation was caused by marked endocytosis of EGF receptors with concomitant ERK attenuation, which stabilizes BIM. These findings demonstrate an unexpected function of an endogenously expressed Akt isoform in promoting - as opposed to suppressing - apoptosis, underscoring that Akt isoforms may exert dissonant functions in malignancy.

Citation Format: Rachel B. Hazan. An Akt3 splice variant lacking the serine 472 phosphorylation site promotes apoptosis and suppresses mammary tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 313.
Abstract 3130: Immune profiling in uterine papillary serous carcinoma cancer patients reveals distinct subpopulations and clinical outcomes
Authors: Vidal Angela Rynne , Lu Karen H. , Mok Samuel C.
Published: August 2018

Uterine papillary serous carcinoma (UPSC) is a rare but aggressive type of endometrial cancer (EC) that represents 10% of cases of EC but accounts for 40% of EC deaths. Patients with UPSC have poor survival rates, a high risk of recurrence and frequently metastasize to the lymph nodes and the omentum. The mechanisms that drive UPSC progression have not been elucidated yet and immune profiling can be a valuable tool to understand the different patient outcomes. In this work, we used formalin fixed paraffin embedded (FFPE) tumor samples from a cohort of 140 patients with UPSC, the largest used to date, to study a panel of 7 immune markers by multiplex immunofluorescence, using the OpalTM system (Perkin Elmer) in the same tissue section. Samples were imaged with Vectra 3.0 multispectral microscope (Perkin Elmer) and images were analyzed using Visiopharm software. Tumor samples from primary, metastatic and recurrent sites were used to study the tumor microenvironment. We observed significant differences in the tumor-infiltrating immune cells between patients that recurred and those that did not. Similarly, immune subpopulations distinguished patients with metastasis compared to those with a less aggressive form of UPSC. Spatial distribution of cytotoxic T cells (CD8+ GranzymeB+) and cancer cells was also found to significantly correlate with overall patient survival. Collectively, our work shows the immune populations play a role in patient outcomes and should be taken into account for future therapeutic strategies.

Citation Format: Angela Rynne Vidal, Karen H. Lu, Samuel C. Mok. Immune profiling in uterine papillary serous carcinoma cancer patients reveals distinct subpopulations and clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3130.
Abstract 3131: Acute myeloid leukemia derived SDF1 repels tumor infiltrating lymphocytes in the bone marrow microenvironment
Authors: Shafat Manar S. , Abdul-Aziz Amina A. , Marlien Christopher , Piddock Rachel , Bowles Kristian M. , Rushworth Stuart A.
Published: August 2018

Introduction: Acute myeloid leukaemia (AML) is an age-related disease with a median age at diagnosis of 71. Combined with co-morbidities, survival of patients with AML above this age bracket is poor with 90% of older adults dying of their disease. Even in younger patients who achieve remission with chemotherapy, relapse is common and occurs from minimal residual disease sequestered in protective niches in the bone marrow microenvironment (BMM). AML as a disease is highly dependent on the bone marrow microenvironment and accordingly it is envisaged that improved outcomes will come from novel treatment strategies derived from an improved understanding of the biology of AML within the bone marrow and their interaction with the immune cells therein.

Objective: Here, we investigate a possible immune evasive mechanism imparted by AML blasts attributed to their localization within the BMM.

Methods and Results: Using RNA-seq data generated from bone marrow derived AML (BM-AML) compared to peripheral blood derived AML (PB-AML) (GEO ID: GSE49642, GSE48846) we identified SDF-1 as the most differentially expressed in favour of BM-AML. Further investigation using matched primary clinical samples (BM verses PB) by real-time PCR analysis and ELISA confirmed the in-silico findings. In addition, primary AML engrafted into NSG mice demonstrated high SDF-1 in the BM derived AML blasts compared to AML blasts derived from the spleen. Lentiviral knock-down of SDF-1 in the AML blasts impaired blast engraftment and prolonged survival of the animals. SDF1-mediated migration of T-cell subsets have been previously identified using varying concentrations of SDF1. We therefore, investigated the migration of CD8+ T cells when co-cultured with AML. We show that CD8+ T-cells migrate away from AML when in culture and this fugetaxis is reduced by the knock-down of SDF1 in the blasts.

Conclusion: We conclude that SDF-1 is secreted by the AML blasts within the BMM contributing to fugetaxis of CD8+ cells away from the blasts. Implications of these finding suggest a possible immune evasive mechanism by the blasts that can be manipulated to potentiate CD8+ infiltration into the AML bone marrow.

Citation Format: Manar S. Shafat, Amina A. Abdul-Aziz, Christopher Marlien, Rachel Piddock, Kristian M. Bowles, Stuart A. Rushworth. Acute myeloid leukemia derived SDF1 repels tumor infiltrating lymphocytes in the bone marrow microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3131.
Abstract 3138: Key role of secreted kinase FAM20C on tumor associated macrophage (TAM) leading to pancreatic cancer progression
Authors: Im Jieun , Lee Yu-Sun , Choi Sun Il , Choi Beom-Kyu , Jeon A-Ra , Park Sang Hyun , Lee Min-Kyeong , Kim Joon-Ki , Kim Yun-Hee
Published: August 2018

FAM20C reported as a novel secreted kinase has the potential of phosphorylation on consensus motif, S-x-E/pS, of secretory proteins or ectodomain of membrane proteins. Numerous substrate candidates through prediction implied FAM20C has the function on tumor microenvironment, however, function and regulatory mechanism of cancer progression by FAM20C has not been defined yet. As tumor associated macrophage (TAM) changes to have the tumor supporting phenotype in response to various environmental stimuli, TAM is the potent regulatory target of FAM20C in tumor microenvironment. In this study, we hypothesized that the secreted kinase FAM20C in tumor microenvironment can support pancreatic cancer progression by regulating TAM contents or polarization. In pancreatic orthotopic xenograft model of FAM20C-overexpressing tumor cells, the tumor growth rate was enhanced and TAM contents (F4/80+/CD11b+/MHCII+) were significantly increased compared to control group, while total macrophage population between two groups had no difference. Moreover, the high level of TAM contents was sustained in the presence of FAM20C till the late stage of tumor progression. In addition, infiltrated tissue macrophages were polarized into TAM by FAM20C treatment Furthermore, increased TAM population by FAM20C suppresses the CD8+ cytotoxic T cell proliferation with anti-tumor function. Collectively, FAM20C might be a key regulatory factor in pancreatic cancer progression by promoting polarization of TAM.

Citation Format: Jieun Im, Yu-Sun Lee, Sun Il Choi, Beom-Kyu Choi, A-Ra Jeon, Sang Hyun Park, Min-Kyeong Lee, Joon-Ki Kim, Yun-Hee Kim. Key role of secreted kinase FAM20C on tumor associated macrophage (TAM) leading to pancreatic cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3138.
Abstract 3139: Innate immune cells expressing myeloperoxidase directly inhibit tumor NF-κB transcriptional activation during tumor initiation
Authors: Liu Tracy W. , Gammon Seth T. , Piwnica-Worms David
Published: August 2018

Neutrophils are considered the primary defenders of the innate immune system. However, in the context of cancer, the role of neutrophils is complex where they can be host protecting or pro-tumorigenic. For example, myeloperoxidase (MPO), the major protein in neutrophils, produces reactive oxygen species (ROS); ROS can lead to tumor regression from cell cytotoxicity or cause DNA-damage resulting in genetic mutations triggering tumor initiation/progression. Using the melanoma cell lines, B16F10, stably expressing a NF-κB-driven Firefly luciferase (FLuc) reporter or an IκBα-FLuc fusion reporter gene, co-culture studies in vitro with bone marrow-isolated-neutrophils from C57BL/6 animals demonstrated inhibition of NF-κB transcriptional activation in a MPO activity-dependent manner. Using bioluminescence imaging with luminol, neutrophils pre-stimulated with 50uM phorbol myristate acetate (PMA) demonstrated an increase in MPO activity by 20.3 ± 2.4 fold over unstimulated neutrophils. When B16F10 reporter cells were co-cultured with PMA pre-stimulated neutrophils, we observed inhibition of IκBα degradation by 33.0% ± 11.9%, resulting in a decrease in NF-κB transcriptional activation compared to co-culture studies with unstimulated neutrophils. The role of neutrophils during B16F10 tumor initiation and growth was compared in C57BL/6 and syngeneic MPO-null mice. Utilizing intravital imaging with skinfold window chamber animal models and imaging reporters, we evaluated in real-time, the recruitment of MPO-expressing cells and tumor NF-κB activation in vivo. Mean NF-κB transcriptional activation within the tumor compartment in MPO-null mice was 42.2% ± 1.6% greater compared to wild type animals. Tumor growth and survival curves demonstrated that B16F10 tumors grew slower in wild-type animals (mean survival of 26.8 days) compared to MPO-null animals (mean survival time of 23.4 days). Using an MPO inhibitor, 4-aminobenzoic acid hydrazide (4-ABAH), we pharmacologically mimicked the MPO-null phenotype in C57BL/6 animals (40mg/kg intraperitoneal injection twice daily). B16F10 tumors grew slower in C57BL/6 animals treated with 4-ABAH (mean survival of 23.8 days) compared to wild type animals. Taken together, these data demonstrated that MPO-expressing cells contribute to host protection during tumor initiation in a NF-κB-dependent manner.

Citation Format: Tracy W. Liu, Seth T. Gammon, David Piwnica-Worms. Innate immune cells expressing myeloperoxidase directly inhibit tumor NF-κB transcriptional activation during tumor initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3139.
Abstract 3143: A novel approach to deplete Treg cells using non-IL-2 blocking anti-CD25-targeting antibodies leads to complete rejection of established tumors
Authors: Solomon Isabelle , Vargas Frederick Arce , Zervas Dimitrios , Qing Chen , Salimu Josephine , Brown Mark , Merchiers Pascal , Boughetane Aghiles , Peggs Karl S. , Goubier Anne , Quezada Sergio A.
Published: August 2018

The accumulation of regulatory T cells (Tregs) in the tumor hampers effector anti-tumor activity and correlates with a bad prognosis in several human cancers. Increasing the effector T cell (Teff) to regulatory T cell (Treg) ratio is known to result in improved control of established tumors. Studies demonstrating high levels of CD25 expression on Tregs but not Teff in human tumors have underscored its relevance as a target for Treg depletion. This supported the development of anti-CD25 depleting monoclonal antibodies (mAbs) as a promising monotherapy as well as combination partner in cancer immunotherapies. To date, anti-CD25 antibodies for clinical use have been designed to deplete CD25 positive cells but also to block IL-2 binding and signaling. Since IL-2 is a critical cytokine involved in T cell activation and proliferation, we hypothesized that a depleting antibody targeting CD25 on Treg cells but not blocking IL-2 signaling on Teff cells would induce a more potent anti-tumor response by depleting Tregs whilst still allowing IL-2 to stimulate effector T cells. We therefore compared the functional activity of anti-mouse CD25 depleting mAbs with and without IL-2 blocking activity. After having confirmed their differential impact on IL-2 signaling, we evaluated the therapeutic activity of these mAbs in various syngeneic tumor models. While both the IL-2 blocking and non-IL-2 blocking mAbs showed equivalent Treg-depleting activity, the antibody sparing IL-2 signaling promoted stronger anti-tumor effect than the IL-2 blocking mAb, with complete tumor regression observed in 70-100% of the mice after a single administration of the antibody. Our data demonstrates that targeting CD25 with ADCC enabled antibodies preserving IL-2 signaling is a novel and powerful strategy for rejection of established tumors via depletion of Treg cells and enhanced, cell intrinsic, IL-2-driven effector T cell activation.

Citation Format: Isabelle Solomon, Frederick Arce Vargas, Dimitrios Zervas, Chen Qing, Josephine Salimu, Mark Brown, Pascal Merchiers, Aghiles Boughetane, Karl S. Peggs, Anne Goubier, Sergio A. Quezada. A novel approach to deplete Treg cells using non-IL-2 blocking anti-CD25-targeting antibodies leads to complete rejection of established tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3143.
Abstract 3145: Characterization of novel immune checkpoint receptors within the breast cancer tumor microenvironment
Authors: Williams Austin D. , Campbell Jean S. , Aicher Lauri D. , Payne Kyle K. , Conejo-Garcia Jose R. , Pierce Robert H. , Tchou Julia
Published: August 2018

Introduction. The interruption of immune checkpoint receptor (ICR)/ligand interaction within the tumor microenvironment has become an important immunotherapy strategy. In breast cancer (BC), neither the ICR phenotype of immune cells of each BC subtype nor the associated immune response is fully characterized. We hypothesize that there are differences in ICR phenotypes between BC subtypes. Methods. To analyze the expression of ICRs (i.e. PDCD1 (PD-1), TIGIT, HAVCR2 (Tim-3), and LAG3 in the tumor microenvironment (TME) of BC, the 956- patient BC cohort of The Cancer Genome Atlas (TCGA) gene expression data was analyzed and normalized against CD3E, a T-cell specific transcript. As validation, we examined various ICR by flow cytometry on 14 matched dissociated breast tumor and peripheral blood mononuclear cells (PBMC) and compared differences across BC subtypes with t-test and ANOVA. In addition, differential gene expression and multispectral analysis were investigated using FFPE specimens and the NanoString and Vectra platforms. Results. TCGA data revealed a higher expression of TIGIT, LAG3 and PD-1 in triple-negative breast cancer (TNBC) when compared to other subtypes (p <0.001, Table 1). PD-L1 (CD274) expression was higher in TNBC than ER/PR+/Her2- cancer (p=-0.008). On flow cytometry, there was a higher co-expression of TIGIT and Tim-3 on CD4 T cells in TNBC than all other subtypes (p=0.003) while ER/PR+Her2- cancers had the highest CD4 expression of TIGIT alone (p=0.04). Compared to ER/PR+Her2- cancers, TNBC had increased expression of transcripts associated with myeloid derived suppressor cells, and decreased expression of transcription factors that regulate lineage specification for CD4 helper T cells. Conclusions. In addition to PD-1, we demonstrated that other ICRs may play significant roles in TME of Her2+ and TNBC BC subtypes. Our results support the development of novel anti-ICR therapy which may synergize with current treatment strategies against these unfavorable BC subtypes.

Citation Format: Austin D. Williams, Jean S. Campbell, Lauri D. Aicher, Kyle K. Payne, Jose R. Conejo-Garcia, Robert H. Pierce, Julia Tchou. Characterization of novel immune checkpoint receptors within the breast cancer tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3145.