bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4654: Hotspot mutations in the core spliceosomal protein SF3B1 promote breast tumorigenesis
Authors: Liu Bo , Ki Michelle , Abdel-Wahab Omar , Chandarlapaty Sarat
Published: August 2019

The core RNA splicing factor SF3B1 is targeted by recurrent hotspot mutations in leukemias and solid tumors with the K700E substitution observed in 2% of unselected breast cancer patients. These mutations promote usage of aberrant intron proximal 3’ splice sites. In breast cancer, the links between these aberrant splicing events and the transformed phenotype are not well understood. Here, we integrated findings from multiple genetically modified model systems and human tumor specimens to understand the contributions of mutant SF3B1 to breast cancer pathogenesis. To identify the aberrant splicing events in breast cancers specifically, we first performed RNA-seq analyses of MCF10A breast epithelial cells with CRISPR knock-in of the K700E mutation. Dysregulation of splicing at over 1,000 splice junctions was discovered, most of which involve the use of alternative 3’ splice sites. Gene expression analysis revealed upregulation of expression of NFκB pathway genes in the SF3B1 mutant cells. SF3B1 mutant cells showed higher phosphorylation of RelA (p65) and, accordingly, greater migration in wound-healing assays. Mis-splicing of exon 5 in MAP3K7, a gene encoding a central regulator of NFκB pathway, significantly reduced MAP3K7 protein expression in SF3B1 K700E mutant cells. Consistent with these data, pharmacologic MAP3K7 inhibition in SF3B1 wild-type breast cells increased phosphorylation of RelA. To further understand the effects of the K700E mutation on breast cancer development and progression, we generated a conditional knock-in mouse model, in which Sf3b1 K700E mutant allele was activated through MMTV promoter-controlled expression of Cre recombinase in mammary epithelium. Accelerated tumor formation was not identified in the mammary gland of these mice. However, RNA-seq analysis of mammary epithelial cells from the Sf3b1 K700E mutant mice revealed dysregulation of alternative splicing including Map3k7. As we found the majority of SF3B1 k700E mutations to co-occur with activating mutations in the PI3K pathway, we additionally introduced the Pik3ca H1047R mutant allele, by mating the MMTV-Cre Sf3b1 K700E mice with Rosa26-Pik3ca H1047R knock-in line. Markedly decreased survival was observed in double mutant animals, which developed mammary tumors faster, compared with single mutant controls. Overall, these data reveal that breast cancer-associated Sf3b1 mutations induce the NFkB pathway to promote invasion and cooperate with the PI3K pathway to accelerate mammary tumor development.

Citation Format: Bo Liu, Michelle Ki, Omar Abdel-Wahab, Sarat Chandarlapaty. Hotspot mutations in the core spliceosomal protein SF3B1 promote breast tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4654.
Abstract 4678: Targeting MEK in EGFR amplified glioma stem like cells induces differentiation
Authors: Park Soon Young , Piao Yuji , Martinez-Ledesma Emmanuel , Dong Jianwen , Khan Sabbir , Mittal Sandeep , Zhang Ze-yan , Sulman Erik P. , Balasubramaniyan Veerakumar , Groot John F. de
Published: August 2019

The median survival for patients with recurrent glioblastoma is nine to twelve months highlighting the need for better therapeutic strategies for this deadly disease. The revolution in cancer genomics has identified multiple amplification events as potential targets for therapeutic intervention in many cancers including glioblastoma. Multiple factors such as incorrect patient selection, inadequate drug delivery across the blood brain barrier, acquired resistance and drug-target heterogeneity may all lead to clinical failure of targeted therapies. Receptor tyrosine kinase (RTK) signaling mediated by EGFR and PDGFR account for the core RTK signaling alterations in glioblastoma. EGFR-amplification (in ~40%) and PDGFR amplification (in ~12%) are detected in receptor tyrosine kinase- dysregulated glioblastoma. The first generation or second-generation EGFR tyrosine kinase small molecule inhibitors failed to show long term therapeutic benefit in glioblastoma patients. Through an unbiased high-throughput screen utilizing our glioma stem-like cells (GSCs) we identified that glioblastoma cells harboring EGFR amplification are uniquely vulnerable to mitogen-activated protein kinase (MEK) inhibitors. MEK inhibition induces apoptosis in EGFR amplified cells at low concentration. Furthermore, RNA sequence analysis of MEK inhibition revealed upregulation of genes related to differentiation in MEK sensitive glioma stem cells. Based upon these in vitro studies we are currently investigating MEK inhibition in a GBM xenograft mouse model. Overall our data suggest that the MEK inhibition could be a potential therapeutic target in a selective group of glioblastoma patients.

Note: This abstract was not presented at the meeting.

Citation Format: Soon Young Park, Yuji Piao, Emmanuel Martinez-Ledesma, Jianwen Dong, Sabbir Khan, Sandeep Mittal, Ze-yan Zhang, Erik P. Sulman, Veerakumar Balasubramaniyan, John F. de Groot. Targeting MEK in EGFR amplified glioma stem like cells induces differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4678.
Abstract 4679: NRF2/SHH signaling cascade promotes tumor-initiating cell lineage and drug resistance in hepatocellular carcinoma
Authors: Leung Hoi Wing , Lau Eunice Yuen Ting , Lee Kin Wah
Published: August 2019

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. The long-term prognosis of HCC remains unsatisfactory due to high recurrence rates and chemoresistance. Solid evidence shows that tumor-initiating cells (T-ICs) are the root of tumor relapse and drug resistance, which led to poor prognosis of patients with hepatocellular carcinoma (HCC). Therefore, identification of crucial transcriptional regulator in liver T-IC may improve the prognosis of these patients. Through in vitro liver T-IC enrichment approach, we identified Nuclear Factor (Erythroid-Derived 2)-Like 2 (NRF2) as transcriptions regulator be significantly upregulated in enriched liver T-IC populations. NRF2 regulated liver T-IC properties including self-renewal, tumorigenicity, and liver T-IC marker expression and significantly affected patients’ clinical outcome. Furthermore, NRF2 repression led to sensitization to doxorubicin and 5-FU treatment. Furthermore, we found that ROS-induced NRF2 activation regulates sorafenib resistance of HCC cells. Mechanistically, NRF2 was found to physically bind to promoter SHH, which triggers activation of sonic hedgehog pathway. NRF2 was significantly correlated with SHH expression, which was associated with poor patient’s survival. The effect of NRF2 knockdown was eliminated upon administration of recombinant SHH, demonstrating that NRF2 mediated the TIC function via upregulation of SHH expression. Our study suggested a novel regulatory mechanism for the canonical sonic hedgehog pathway that may function through NRF2/SHH/GLI signaling axis, thus mediating T-IC phenotypes. Targeting NRF2 mediated signaling cascade alone or in combination with other treatment modalities may be a new a potential therapeutic approach for treatment of HCC.

Citation Format: Hoi Wing Leung, Eunice Yuen Ting Lau, Kin Wah Lee. NRF2/SHH signaling cascade promotes tumor-initiating cell lineage and drug resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4679.
Abstract 4706: Temperature-dependent transcription artifacts and cell population biases in scRNAseq data are minimized by tissue dissociation at low temperatures
Authors: O'Flanagan Ciara H. , Campbell Kieran R. , Kabeer Farhia , Zhang Allen , Lim Jamie , Shah Sohrab P. , Aparicio Samuel
Published: August 2019

Single cell RNA sequencing (scRNAseq) is a powerful tool, particularly for studying complex biological systems, such as tumor heterogeneity and the tumor microenvironment, which may not be resolved by sequencing of bulk material. Nonetheless, it is not without limitations, which include the technical challenges of generating a high quality single cell suspension. Dissociation of tissue to single cell suspension requires mechanical and enzymatic disruption, and the effect of these methods on gene expression or cellular population bias has not been established. In this study, we examined the effects of enzymatic dissociation on cell population capture and transcriptional changes at single cell resolution in breast and ovarian cancer patient samples, patient-derived breast cancer xenografts and cultured cell lines. scRNAseq data showed that enzymatic dissociation of tissues at 37oC with collagenase resulted in significant induction of heat shock, stress and immediate response genes, which was conserved across all tissues. This gene expression induction was not observed when tissues were dissociated at 6oC with a protease derived from a Himalayan glacier soil bacterium. Moreover, dissociation of patient tumors at low temperature enhanced the abundance of rare cell populations, including B-cells, T-Cells and cytotoxic T-cells, which were significantly depleted following dissociation at 37oC. These biases resulting from standard sample preparation methods could significantly affect biological interpretation of scRNAseq data, and can be minimized by dissociation of tissues at low temperature.

Note: This abstract was not presented at the meeting.

Citation Format: Ciara H. O'Flanagan, Kieran R. Campbell, Farhia Kabeer, Allen Zhang, Jamie Lim, Sohrab P. Shah, Samuel Aparicio. Temperature-dependent transcription artifacts and cell population biases in scRNAseq data are minimized by tissue dissociation at low temperatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4706.
Abstract 4711: PRMT5 selective inhibitor enhances therapeutic efficacy of cisplatin in lung adenocarcinoma cells
Authors: Bajbouj Khuloud , Ramakrishnan Rakhee , M.H.Ihmaid Ahmed , Ali Suhaib Al Haj , Alalool Abdulla , Abdullah Reem , Saber-Ayad Maha , Hamid Qutayba
Published: August 2019

Background: Lung cancer is the most common cancer globally, with an estimate of more than 2 million new cases every year. Protein arginine methyltransferase 5 (PRMT5) is an enzyme that is identified to be involved in multiple cellular events, including gene expression regulation transcriptional regulation and cell division. It has been shown that PRMT5 is highly expressed in cancers including lung adenocarcinoma, hepatocellular carcinoma, and melanoma, raising evidence that PRMT5 might be involved in tumorigenesis. The aim of this study is to examine the potential selective anti-neoplastic activity of AMI-1 and cisplatin on lung adenocarcinoma.

Experimental methods: The effect of AMI-1 and subsequent PRMT5 activity inhibition on lung adenocarcinoma (A549 cell line) behavior with the standard chemotherapeutic agent, cisplatin was assessed, by measuring cell viability using MTT assay, PMRT5/β-catenin protein levels via Western Blotting, the extent of cell migration through wound healing assay, and survival of cancer cells by performing cell cycle progression and Annexin-V staining assays along with both drugs combination potential toxicity that was evaluated in human bronchial epithelial cells (HBEC).

Results and Discussions: Combination treatment with 10 µM AMI-1 and IC50 of Cisplatin (23.4 µM) significantly reduced viable cell percentages at 24 and 48 hours. Treatment with both drugs at 48 hours led to a decreased cells migration rate. G2/M arrest in A549 cells was evident at 24 hours of AMI-1 addition, which was reinforced after drugs combined treatment. Furthermore, 48 hours of both drug administration hindered the ability of A549 cells to recycle again as they G1 occupying cells percentage was increased after 48 hours of combination treatment. There was a minor cell death induction after 48 hours of treatment with both drugs. Western blot analysis showed that there was no evidence change in total PRMT5 and β-catenin protein levels. Nevertheless, there was a reduction in demethylation Histone 4, a PRMT5- downstream target, after 48 hours treatment with AMI-1 alone or in combination with Cisplatin. Interestingly, as combination approach increased anti-survival effect on lung cancer cells, it exhibited cyto-protective abilities on normal epithelial cells.

Conclusion: This study shows a novel selective antitumor activity of AMI-1 and cisplatin in adenocarcinoma cells.

Citation Format: Khuloud Bajbouj, Rakhee Ramakrishnan, Ahmed M.H.Ihmaid, Suhaib Al Haj Ali, Abdulla Alalool, Reem Abdullah, Maha Saber-Ayad, Qutayba Hamid. PRMT5 selective inhibitor enhances therapeutic efficacy of cisplatin in lung adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4711.
Abstract 4713: Extraction, purification and evaluation of prmt5-inhibitory phytochemical compounds for the treatment of prostate adenocarcinoma
Authors: Richmond Oliver H. , Wang Zhengxin
Published: August 2019

The development and advancement of prostate cancer is supported by a plethora of genetic and proteomic abnormalities, including events of post-translational modifications. The protein arginine methyltransferase 5 (PRMT5) enzyme regulates epigenetic events of histone modifications and of post-translational modifications within protein signaling pathways. Under abnormal conditions of overexpression and/or upregulation, PRMT5 functions by constitutively driving the growth and/or proliferation of dysregulated cells. Overexpression and/or upregulation of PRMT5 correlates with disease progression as observed among numerous cancer types, including breast, colorectal, leukemia, lung, melanoma and prostate cancers. We demonstrated previously that PRMT5 localized to the cytoplasm, functions to drive both growth and proliferation of prostatic tumors. Plants naturally produce chemical toxins as mechanisms of defense against microbial and other biological threats. Human exploitation, consumption and/or application of agents isolated from plants for therapeutic intervention dates back throughout the millennia. In this study, we extracted, purified and evaluated natural, small, chemical compounds from plant products that antagonize PRMT5 activity in prostate cancer cells. We found that crude and purified extracts of Dendrobium, Fennel Seed and Magnolia plants attenuated prostate tumor growth and proliferation by selective inhibition of PRMT5 methyltransferase activity. These findings establish the first set of natural PRMT5-specific inhibitors reported.

Citation Format: Oliver H. Richmond, Zhengxin Wang. Extraction, purification and evaluation of prmt5-inhibitory phytochemical compounds for the treatment of prostate adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4713.
Abstract 4725: Efficacy of a new small-molecule inhibitor of histone deacetylase 6 (HDAC6) in preclinical models of B-cell lymphoma and acute myeloid leukemia
Authors: Villanueva Lorea , Perez-Salvia Montserrat , Aldaba Eneko , Vara Yosu , Fabre Myriam , Ferrer Cristina , Masdeu Carme , Zubia Aizpea , Sebastian Eider San , Otaegui Dorleta , Llinàs-Arias Pere , Rosselló-Tortella Margalida , Berdasco María , Setien Fernando , Moutinho Catia , Villanueva Alberto , González-Barca Eva , Muncunill Josep , Navarro José Tomás , Piris Miguel Ángel , Cossio Fernando , Esteller Manel
Published: August 2019

Histone deacetylase 6 (HDAC6) is a protein modifier that is an increasingly attractive pharmacological target. Interestingly, the observation that the HDAC6 knock-out mouse is not lethal, in contrast to those undergoing complete loss of class I, II and III HDACs, suggests that specific HDAC6 inhibitors may be better tolerated than pan-HDAC inhibitors or drugs that target the other HDAC classes. In this regard, the compound ACY-1215 (Rocilinostat), the described selective HDAC6 inhibitor, is undergoing clinical trials for the treatment of multiple myeloma. Taking into account the previous information about HDAC6 inhibitor structures, the structural differences between HDAC6 and other HDAC isoforms and also the structural information of other developed HDAC inhibitors, we have previously designed and synthesized a new potential HDAC6 selective inhibitor, QTX125 with growth inhibitory effects in mantle cell lymphoma (MCL) cell lines, mouse models and ex vivo treatment of primary samples obtained from patients with MCL. Herein, we have extended these findings to show that the newly identified HDAC6 inhibitor QTX125 is also able to inhibit the growth of preclinical models of other B-cell lymphomas such as follicular lymphoma and Burkitt’s cell lymphoma, but also of acute acute myeloid leukemia. In addition beyond a-tubulin, a well known HDAC6 target, we have developed a pharmacological and proteomic screening to identify other proteins modified by HDAC6 that can contribute to the described lymphoma and leukemia phenotypes.

Citation Format: Lorea Villanueva, Montserrat Perez-Salvia, Eneko Aldaba, Yosu Vara, Myriam Fabre, Cristina Ferrer, Carme Masdeu, Aizpea Zubia, Eider San Sebastian, Dorleta Otaegui, Pere Llinàs-Arias, Margalida Rosselló-Tortella, María Berdasco, Fernando Setien, Catia Moutinho, Alberto Villanueva, Eva González-Barca, Josep Muncunill, José Tomás Navarro, Miguel Ángel Piris, Fernando Cossio, Manel Esteller. Efficacy of a new small-molecule inhibitor of histone deacetylase 6 (HDAC6) in preclinical models of B-cell lymphoma and acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4725.
Abstract 4745: Reversal by inhibiting XBP1 activation of glucose deprivation-induced tolerance to multiple anticancer therapies
Authors: Jeong Mini , Jo Serin , Park Serkin , Sohn Jeongwon , Park Yun Gyu
Published: August 2019

Tumor microenvironment is harsh condition including low pH, hypoxia and nutrient deprivation. Cancer cells can switch their phenotypes and characteristics dependent on the microenvironment. Most previous researches have focused mainly on the effect of hypoxia on cancer cells plasticity and resistance to anticancer therapies. Despite the accumulation of emerging evidences, the effect of nutrient deficiency to multiple anticancer therapies is still unclear.In this study, human lung adenocarcinoma cells (A549), triple-negative breast cancer (MDA-MB-231), hepatocellular carcinoma cells (HepG2), pancreas adenocarcinoma cells (AsPC-1) were cultured in the medium containing low concentrations of glucose. Low sensitivity to cisplatin or taxol was observed in the cancer cells maintained for 28 weeks or so under the glucose-deprived condition. In addition, the multidrug tolerant cells were also insensitive to cytotoxic T cell (CTL)-based immunotherapy and had increased migration ability compared with control group under normal glucose condition. Reversion to normal glucose condition only for short term period (at least 4 days) made the multidrug tolerant cells susceptible to cisplatin-induced apoptotic cell death. In the unfolded protein response, BiP-XBP1 signaling was increased but PERK signaling was decreased in response to long-term glucose deprivation. Sensitivity to cisplatin treatment and CTL-based immunotherapy was restored by inhibiting XBP1 splicing in glucose deprivation-induced multidrug tolerant cells in various types of cancer.This study demonstrated that glucose deprivation-induced tolerance to multiple anti-cancer therapies is transient and reversible, and requires activation of IRE1-XBP1 pathway.This research was supported by the National Research Foundation of Korea (2018R1D1A1B07045297, 2016R1D1A1A02936945 and 2017R1D1A1B03036008).

Citation Format: Mini Jeong, Serin Jo, Serkin Park, Jeongwon Sohn, Yun Gyu Park. Reversal by inhibiting XBP1 activation of glucose deprivation-induced tolerance to multiple anticancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4745.
Abstract 4746: Identifying states of collateral sensitivity during the evolution of therapy resistance in Ewing’s sarcoma
Authors: McClure Erin , Hitomi Masahiro , Scarborough Jessica , Scott Jacob
Published: August 2019

During the evolution of therapeutic resistance many cancers exhibit a phenomenon termed collateral sensitivity, where resistance to one drug aligns with sensitivity to another drug. It has further been shown, in leukemia and non-small cell lung cancer, that this state of collateral sensitivity can vary over time. We hypothesized that this process will also occur in Ewing’s sarcoma (EWS), and that these phenotypic states can be identified with molecular signatures based on gene expression. The standard chemotherapy for EWS is repeated cycles of two alternating drug cassettes, one is a combination of vincristine, doxorubicin, and cyclophosphamide (VDC), and a second is a combination of ifosfamide and etoposide (IE). This treatment is highly effective, though advanced stage disease often relapses, and then drug resistance invariably develops, a process following the laws of Darwinian evolution. Finding predictable patterns, or signatures, of collateral sensitivity would provide a useful decision support tool in the clinic to help guide chemotherapy choices. Recent studies in other cancers, bacteria, and in silico simulations have explored and identified optimal timing of switching among the drugs of collateral sensitivity to achieving maximal efficacy. To analyze temporal changes in collateral sensitivity in EWS, we exposed two EWS cell lines, A673 and TTC-466, to half maximal inhibitory concentrations of VDC/IE combinations in vitro. As the cultures recovered from drug exposures, we determined their sensitivities against a panel of drugs including the ones used for the treatment and a new generation of drugs that show efficacy against EWS. The new drugs consist of SP2509, pazopanib, olaparib, dactinomycin, temozolomide, vorinostat, and SN-38. We also collected RNA samples to analyze the transcriptomic alterations which correlate with changes in their collateral sensitivity. Significant findings for the A673 cell line include increased resistance to dactinomycin, doxorubicin, etoposide and vincristine for all treatment replicates, and maximal sensitivity to temozolomide halfway through the treatment schedule for all treatment replicates. Notably, every tumor is different and may develop collateral sensitivity at unique time points. Even in this experiment we observe variation between evolutionary replicates that were originally genetically identical. Future work should include investigating a quick and cost-effective method for testing temporal collateral sensitivity of individual tumors so our findings can be implemented clinically.

Citation Format: Erin McClure, Masahiro Hitomi, Jessica Scarborough, Jacob Scott. Identifying states of collateral sensitivity during the evolution of therapy resistance in Ewing’s sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4746.
Abstract 477: Targeted deep sequencing contributes to guiding personalized targeted therapy in advanced biliary tract cancer
Authors: Feng Feiling , Cheng Qingbao , Zhang Dadong , Li Bin , Qin Hao , Xu Chang , Han Miao , Yu Yong , Li Zhizhen , Li Jing-Yu , Qiu Zhiquan , Xiong Lei , Liu Chen , Li Fugen , Yi Bin , Jiang Xiaoqing
Published: August 2019

Purpose: Biliary tract cancer (BTC) is a devastating disease of the digestive system with poor prognosis. Targeted therapy based on specific genetic alterations has been proven to be an effective treatment for some cancer subtypes. However, the effect of this targeted therapy is unclear in BTC. In this study, we aimed to explore the clinical efficacy and safety of personalized targeted therapy guided by targeted deep sequencing for advanced biliary tract cancer (BTC) patients.

Materials and Methods: In this retrospective study, targeted deep sequencing was employed for 49 patients with BTC and the recommendation of biologic agent was offered. Among 32 patients with stage IV and R2 resection, 21 patients underwent conventional chemotherapy (mGEMOX) (chemotherapy group), while the remaining 11 patients received a personalized targeted agent (targeted therapy group). The progression-free survival (PFS), overall survival (OS), disease control rate (DCR) were used to assess the efficacy of treatments, while the grade of treatment-related toxicities was evaluated for safety.

Results: The genomic landscape of 49 patients with BTC was depicted by targeted deep sequencing. Further analysis of all alterations demonstrated that these altered genes were highly enriched in the ERBB family or cell cycle pathway. After a median follow-up of 12 months, the targeted therapy group had a significant prolonged PFS (4.5 months vs. 1.5 months, P = 0.014) and a trend of prolonged OS (12.9 months vs. 4.1 months, P = 0.104) in comparison to the chemotherapy group. The DCR in the targeted therapy group was marginally higher but without statistical significance (63.6% vs. 33.3%, P = 0.142). In addition, there was no statistically significant difference in the percentage of patients that experienced Grade >2 treatment-related toxicities in either treatment group (36.4% vs 19.0%, P = 0.397).

Conclusions: This real-world clinical study suggests that targeted deep sequencing contributes to guiding personalized targeted therapy based on individual actionable mutations, which may benefit advanced BTC patients. Large umbrella trials of personalized precision therapy are needed to further confirm the clinical efficacy and safety of this therapeutic strategy in BTC.

Note: This abstract was not presented at the meeting.

Citation Format: Feiling Feng, Qingbao Cheng, Dadong Zhang, Bin Li, Hao Qin, Chang Xu, Miao Han, Yong Yu, Zhizhen Li, Jing-Yu Li, Zhiquan Qiu, Lei Xiong, Chen Liu, Fugen Li, Bin Yi, Xiaoqing Jiang. Targeted deep sequencing contributes to guiding personalized targeted therapy in advanced biliary tract cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 477.
Abstract 4776: Characterizing an oncolytic adenovirus modified with the CXCL12 ligand for breast cancer therapy
Authors: O'Bryan Samia M. , Mathis J. Michael
Published: August 2019

Breast cancer is the most commonly diagnosed cancer in women, making up nearly 30% of all diagnosed cancer cases each year. While localized breast cancer is easily treated, advanced cases are difficult to treat resulting in poor five-year survival rates. Thus, new therapies capable of increasing treatment efficacy are needed. Oncolytic virotherapy using human adenovirus is a novel therapeutic approach capable of specifically targeting cancer cells while sparing normal tissue. The adenovirus vector is well-characterized and is uniquely suited for oncolytic virotherapy due to its systemic stability, good safety profile and the ability to infect a broad range of dividing and non-dividing cells. Replication of oncolytic adenoviruses within cancer cells causes the lysis of the cells and subsequent spread of progeny virions within the surrounding tumor stroma. One challenge in targeting cancer cells with adenovirus vectors has been the low expression of the endogenous receptor, the Coxsackie and adenovirus receptor (CXADR), prompting the search for new receptor targets. In this study, we engineered an adenovirus to contain the CXCL12 ligand to target the chemokine receptors CXCR4 and CXCR7 overexpressed in a variety of tumors. Altered expression of these receptors drives tumor progression, migration, invasion and metastasis. Previously, we developed a bispecific adaptor molecule containing the CXCL12 ligand to redirect a replication-deficient wild-type adenovirus to breast cancer cells overexpressing CXCR4. In the current study, we engineered a replication-competent oncolytic adenovirus modified with the CXCL12 ligand to target cancer cells overexpressing CXCR4 and CXCR7. A recombinant gene was constructed using the tail domain of the adenovirus fiber gene fused to the trimerization domain of the T4 fibritin gene followed by the mature CXCL12 sequence. After viral production, the presence of a modified fiber and the CXCL12 ligand were confirmed. Subsequently, the vector was tested in a panel of breast cancer cells for infection and cell killing efficacy in vitro. Receptor knock down and overexpression studies confirmed the specificity of the virus. Together, these studies address the hypothesis that constructing a retargeted oncolytic adenovirus using the CXCL12 ligand provides selective infection and killing of breast cancer cells overexpressing CXCR4 and CXCR7. These results support the rationale for further development of retargeted adenovirus vectors for oncolytic virotherapy in patients. Future experiments will be conducted to assess tumor targeting using a xenograft mouse model of human breast cancer.

Citation Format: Samia M. O'Bryan, J. Michael Mathis. Characterizing an oncolytic adenovirus modified with the CXCL12 ligand for breast cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4776.
Abstract 4777: Anti-tumor effect of adenovirus encoding APE1/Ref-1 in breast cancer xenografts: anti-inflammation and apoptotic cell death
Authors: Choi Sunga , Lee Yu Ran , Kim Yeon Hyang , Kim Ki Mo , Park Myoung Soo , Jeon Byeong Hwa
Published: August 2019

In persistent inflammatory state involving the chronic activation of the immune system, the interplay between breast cancer and stromal cells is closely associated. We have previously shown that APE1/Ref-1 which could inhibit inflammatory signaling by reductive conformational change of cytokines receptors. In response to hyperacetylation, stimulation by secreted Ac-APE1/Ref-1 binding to RAGE initiated apoptotic cell death in TNBC models, in vitro and in vivo. In the present study, we used Ad-APE1/Ref-1 adenovirus, which can be overexpressed and be secreted into the blood of xenografts. We investigated how the Ad-APE1/Ref-1 influences growth retardation and apoptotic cell death of inflammation associated TNBC in vivo. The Ad-APE1/Ref-1-injection in MDA-MB 231 orthotopic xenografts caused a decrease in the volume and weight of tumors as shown in IVIS images. The treatment of Ad-APE1/Ref-1 also induced an inhibition of tumor growth and development, leading to apoptotic cell death, accompanied by generation of reactive oxygen species. Tumor tissues derived from Ad-APE1/Ref-1 exhibited apoptotic bodies compared with tumors of control or Adβ-galactosidase-injected mice. Moreover, level of inflammatory cytokines was evaluated in plasma of the xenografts; ratio of anti- to pro inflammatory cytokines was comparable with one of normal mice. The PAK1-STAT-3-NFkB axis in inflammatory signaling of tumor tissues derived from AdAPE1/Ref-1 injected mice was significantly inhibited compared to those of control or Adβ-galactosidase-injected mice. These results suggest that regulation of inflammatory signaling with adenoviral mediated APE1/Ref-1 in tumors of MDA-MB-231 xenografts modulates cytokine secretion, thereby inhibiting cancer cell growth.

Note: This abstract was not presented at the meeting.

Citation Format: Sunga Choi, Yu Ran Lee, Yeon Hyang Kim, Ki Mo Kim, Myoung Soo Park, Byeong Hwa Jeon. Anti-tumor effect of adenovirus encoding APE1/Ref-1 in breast cancer xenografts: anti-inflammation and apoptotic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4777.
Abstract 4781: Preclinical assessment of efficacy and safety of novel oncolytic adenovirus for therapy of disseminated lung cancer
Authors: Atasheva Svetlana , Yao Jia , Young Cedrick , Paolo Nelson C. Di , Wyche Henry , Shayakhmetov Dmitry M.
Published: August 2019

The majority of lung cancer patients do not respond to current immune-oncology drugs due to low PD1 and PD-L1 expression on immune and cancer cells. Oncolytic viruses are being actively explored as an alternate modality for cancer therapy due to their natural ability to kill infected cells through viral replication. In this study, we analyzed the anti-tumor efficacy and safety profile of AVID-317 oncolytic virus in pre-clinical mouse models of disseminated lung cancer. AVID-317 is a novel oncolytic adenovirus possessing a set of mutations in the virus capsid, which ablate virus interactions with cellular b3 integrins on macrophages and hepatic cells, resulting in virus de-targeting from the liver after intravenous administration. In vitro studies demonstrated that 70% of tested human non-small cell lung cancer (NSCLC)-derived cell lines (N=16) are sensitive to AVID-317 infection, and 85% of AVID-317-sensitive NSCLC lines express no or low PD-L1. Intravenous injection of control parental human adenovirus type 5 (HAdv5) at a dose of 1e11 v.p. per mouse into wild-type mice resulted in release of pro-inflammatory cytokines IL-1b, IL-6, IL-16, and G-CSF into the bloodstream at 6 hours post virus administration. Furthermore, this dose of HAdv5 was lethal due to severe hepatotoxicity within 48 h post virus injection. In contrast, intravenous administration of AVID-317 in the range of doses up to 1e12 v. p. per mouse resulted in no detectible IL-6 and significantly reduced IL-1b, IL-16, G-CSF amounts in the blood. Importantly, AVID-317 administration did not result in mortality. Next, we grafted PD-L1-low human adenocarcinoma A549-Luc-C8 cells into lungs of NCr nude mice to establish an orthotopic model of disseminated lung cancer. When tumor burden reached between 2xe6 to 8e6 RLU, mice were enrolled in four cohorts and treated with AVID-317 at doses of 5e10, 1e11, or 3e11 v.p. per mouse or administered with saline only (control group; N=>10 in each cohort). Mice were euthanized upon detection of 20% weight loss compared to baseline. The median survival in saline-treated group was 57 days post treatment. However, median survival has not be reached for AVID-317-treated groups for the duration of the study (80 days). Taken together, our study demonstrates feasibility of using AVID-317 oncolytic adenovirus for systemic therapy of disseminated lung cancer and its greatly improved safety profile observed upon intravenous virus delivery.

Citation Format: Svetlana Atasheva, Jia Yao, Cedrick Young, Nelson C. Di Paolo, Henry Wyche, Dmitry M. Shayakhmetov. Preclinical assessment of efficacy and safety of novel oncolytic adenovirus for therapy of disseminated lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4781.
Abstract 4787: New and potent small molecule as EF2K inhibitor: A novel EF2K inhibitor
Authors: Onder Ferah Comert , Ay Mehmet , Durdagi Serdar , Ozpolat Bulent , Kantarcioglu isik
Published: August 2019

Triple-negative breast cancer (TNBC) which constitutes 15-20% of breast cancers, is the most aggressive and chemo-resistant subtype of breast cancer and associated with the highest mortality rates of mortality among all breast cancer subtypes. TNBC is characterized by a lack of molecular targets (e.g., estrogen, progesterone, and HER2 receptors. Current treatment options for TNBC are still limited to conventional chemotherapies such as anthracyclines (e.g., doxorubicin) and taxane-based therapeutics. Identification of new molecular targets is critical for development of novel targeted treatments to improve poor patient prognosis and survival. We recently found that expression of eukaryotic elongation factor-2 kinase (EF2K), an unusual alpha kinase, is commonly upregulated in the majority of TNBC patients and associated with poor prognosis. We showed that EF2K is a potential molecular driver by promoting cell proliferation motility and invasion and drug resistance and validated it as a molecular target by RNA based therapeutic (i.e, siRNA and microRNA) in various TNBC models in mice (Tekedereli 2012, Hamurcu 2017, Bayraktar 2017). Several inhibitors of eEF2K have been described in the previous studies. However, these inhibitors have been neither specific nor potent. Small molecule inhibitor was synthesized in many steps by using different synthesis methods and also performed all of the structural analysis (FT-IR, 1H-NMR, 13C-NMR and LC-MS-MS). Computational studies were performed by using Schrödinger 2015 programme to investigate of in vitro activity of the compounds. By performing in vitro assays we identified a potent inhibitor of EF2K which inhibited p-EF2 at 1 μM (2 hours) detected by Western blots. Treatment of TNBC cells with this compound starts to inhibits cell proliferation and colony formation at 5-10 μM concentrations. The further studies are still ongoing. In conclusion, our results suggest that our newly designed small molecule may be potent EF2K inhibitor and can be used for targeting EF2K in TNBC.

Note: This abstract was not presented at the meeting.

Citation Format: Ferah Comert Onder, Mehmet Ay, Serdar Durdagi, Bulent Ozpolat, isik Kantarcioglu. New and potent small molecule as EF2K inhibitor: A novel EF2K inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4787.
Abstract 479: BRAF and KRAS mutation define distinct subtypes of the CpG island methylator phenotype in colorectal cancers
Authors: Whitehall Vicki L. , Fennell Lochlan , Dumenil Troy , Hartel Gunter , Nones Katia , Bond Catherine , McKeone Diane , Patch Ann-Marie , Kazakoff Stephen , Pearson John , Waddell Nicola , Wirapati Pratyaksha , Lochead Paul , Ogino Shuji , Tejpar Sabine , Leggett Barbara
Published: August 2019

BACKGROUND AND AIMS: Colorectal cancer is an epigenetically heterogeneous disease, however the extent and spectrum of the CpG Island Methylator Phenotype (CIMP) is not clear.METHODS: Genome scale methylation and transcript expression were measured using the Illumina HM450 DNA methylation and HT12 V3 expression microarrays in 216 unselected colorectal cancers. Mutations in epigenetic regulators were assessed using CIMP-classified Cancer Genome Atlas exomes.RESULTS: CIMP-High cancers dichotomised into CIMP-H1 and CIMP-H2 based on methylation profile, which was supported by over-representation of BRAF (74%, P<0.0001) or KRAS (55%, P<0.0001) mutation. Congruent with increasing methylation, there was a stepwise increase in patient age from 62 years in the CIMP-Negative subgroup to 75 years in the CIMP-H1 subgroup (P<0.0001). CIMP-H1 were predominantly comprised of consensus molecular subtype 1 (CMS1) cancers (70%) whilst CMS3 was over-represented in the CIMP-H2 subgroup (55%). PRC2-marked loci were subjected to significant gene body methylation in CIMP cancers (P<1.6x10-78). We identified oncogenes susceptible to gene body methylation and Wnt pathway antagonists resistant to gene body methylation. CIMP cluster specific mutations were observed for in chromatin remodeling genes, such as in the SWI/SNF and NuRD complexes, suggesting synthetic lethality.CONCLUSION: There are five clinically and molecularly distinct subgroups of colorectal cancer. We show a striking association between CIMP and age, gender and tumor location and identify an unidentified role for gene body methylation in progression of serrated neoplasia. These data support our recent findings that CIMP is uncommon in young patients and that BRAF mutant polyps in young patients may have limited potential for malignant progression.

Citation Format: Vicki L. Whitehall, Lochlan Fennell, Troy Dumenil, Gunter Hartel, Katia Nones, Catherine Bond, Diane McKeone, Ann-Marie Patch, Stephen Kazakoff, John Pearson, Nicola Waddell, Pratyaksha Wirapati, Paul Lochead, Shuji Ogino, Sabine Tejpar, Barbara Leggett. BRAF and KRAS mutation define distinct subtypes of the CpG island methylator phenotype in colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 479.
Abstract 48: Cellular dynamics and heterogeneity of active Kras induced-gastric pre-neoplasia in mouse
Authors: Choi Eunyoung , Vega Paige N. , Min Jimin , Engevik Amy C. , Williams janice A. , Yang Qing , Patterson Loraine M. , Simmons Alan J. , Lau Ken S. , Magness Scott T. , Goldenring James R.
Published: August 2019

Cellular heterogeneity in cancer development is considered as a source of cancer cell generation and drug resistance. However, cellular heterogeneity has not been fully eluciated during metaplasia progression to gastric pre-neoplasia and neoplasia in gastric carcinogenesis. Recent investigations have suggested that Ras activation, rather than Ras mutation, may promote the gastric carcinogenesis. We have recently established several in vitro metaplastic/pre-neoplastic organoid lines; Meta3-metaplasia and Meta4-pre-neoplasia, derived from mouse stomachs induced active Kras expression in chief cells and investigated cellular heterogeneity and plasticity that may lead to the progression of metaplasia to gastric neoplasia. While both Meta3 and Meta4 initially formed spherical or spheroidal structures and generated complicated budding or cyst-like structures between 2 and 4 weeks, Meta4 cells displayed more disorganized structures and aggressive behaviors than Meta3. We also examined altered cellular behaviors, structural changes and survival of Meta4 organoids after MEK inhibition which is a downstream mediator of Kras signaling pathway. The Meta4 treated with Selumetinib for 3 days died or did not show an increase in size at various concentrations. However, some Meta4 organoids showed an ability to survive and retained a spheroidal structure despite the Selumetinib treatment. Single cell-RNA sequencing (scRNA-seq) data analysis demonstrated distinct characteristics of Meta4 cells as pre-neoplastic cells and a signature of presence and heterogeneity of pre-neoplasia stem cell (PNSC) populations. The scRNA-seq data analysis also revealed that cellular behavior and dynamics of pre-neoplastic cells can be controlled by MEK inhibition, but the PNSC populations still maintain the niche for stem-like cells in gastric pre-neoplasia. The isolated two PNSC populations showed functional differences and the stemness of the PNSCs in pre-neoplastic stage through high-throughput functional analysis. Therefore, our study concludes that putative gastric cancer stem cells in gastric pre-neoplasia might be the source for maintenance of heterogeneity and contribute to the progression of gastric pre-neoplasia to cancer.

Citation Format: Eunyoung Choi, Paige N. Vega, Jimin Min, Amy C. Engevik, janice A. Williams, Qing Yang, Loraine M. Patterson, Alan J. Simmons, Ken S. Lau, Scott T. Magness, James R. Goldenring. Cellular dynamics and heterogeneity of active Kras induced-gastric pre-neoplasia in mouse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 48.
Abstract 4819: Sacituzumab Govitecan (IMMU-132) in uterine serous carcinoma
Authors: Lopez Salvatore , Han Chanhee , Zeybek Burak , Bonazzoli Elena , Bianchi Anna , Manara Paola , Bellone Stefania , Manzano Aranzazu , Perrone Emanuele , Zammataro Luca , Altwerger Gary , Santin Alessandro D.
Published: August 2019

Objective: Uterine serous carcinoma (USC) is an aggressive variant of endometrial cancer with poor prognosis. Sacituzumab govitecan (IMMU-132) is a novel antibody-drug conjugate (ADC) targeting trophoblast antigen 2 (Trop-2), a cell surface glycoprotein highly expressed in many epithelial tumors including USC, to deliver SN-38, the active metabolite of irinotecan. The objective of this study was to preclinically evaluate the efficacy of IMMU-132 against primary USC cell lines and xenografts.

Methods: Trop-2 expression in primary tumor cell lines and USC cell viability after exposure to IMMU-132 ADC (hRS7-CL2A-SN-38), non-targeting control ADC (h679-CL2A-SN-38), and naked antibody hRS7 IgG were evaluated using RT-PCR and flow-cytometry-based-assays. Antibody-dependent-cell-cytotoxicity (ADCC) against Trop-2+ and Trop-2- USC cell lines was evaluated in vitro using 4 hr Chromium release assays. Finally, in vivo activity of Sacituzumab govitecan was tested against Trop-2+ USC xenografts by 3 twice-a-week retro-orbital injection of 500 μg of IMMU-132, control-ADC, and hRS7 naked-IgG.

Results: Overexpression of Trop-2 was detected in 67% (8 out of 12) of primary USC cell lines. Primary tumors overexpressing Trop-2 were significantly more sensitive (ie, lower IC50) to IMMU-132 (hRS7-CL2A-SN-38) when compared to control ADC (h679-CL2A-SN-38). Both sacituzumab govitecan (IMMU-132) and the naked antibody hRS7 induced high level of ADCC against Trop2+ USC cell lines while no cytotoxicity was detected against Trop-2 negative tumors. In vivo experiments comparing IMMU-132 activity to control ADC and hRS7 showed a dramatically improved tumor suppression and increased survival in IMMU-132 treated mice when compared to controls (P = 0.0001 and P = 0.0002, respectively).

Conclusion: IMMU-132 demonstrated remarkable antitumor activity against biologically aggressive USC overexpressing Trop-2. Our preclinical results combined with the dramatic clinical response recently reported in an USC patient treated with IMMU-132 (https://doi.org/10.1016/j.gore.2018.05.009) strongly supports further clinical development of sacituzumab govitecan in USC patients with advanced/recurrent disease. (ie, clinical trial IND 140394). Furthermore, due to a cleavable linker that can cause bystander effect, sacituzumab govitecan could be also effective in tumors with heterogenous TROP-2 expression.

Citation Format: Salvatore Lopez, Chanhee Han, Burak Zeybek, Elena Bonazzoli, Anna Bianchi, Paola Manara, Stefania Bellone, Aranzazu Manzano, Emanuele Perrone, Luca Zammataro, Gary Altwerger, Alessandro D. Santin. Sacituzumab Govitecan (IMMU-132) in uterine serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4819.