bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract CN07-01: Cellular plasticity in cancer: Driving force and therapeutic target
Authors: Brabletz Thomas
Published: August 2018

We have shown that in particular tumor cells at the invasive front undergo a partial epithelial-mesenchymal transition (EMT) and aberrantly express EMT-associated transcription factors (EMT-TFs). The amount of such cancer cells strongly correlates with metastasis formation and poor clinical outcome in human cancers. Strikingly, metastases show a mesenchymal-epithelial retransition (MET) with a redifferentiated phenotype, indicating high cancer cell plasticity and supporting a regulatory role of the tumor environment. We described that the EMT-TF ZEB1 is a crucial determinant of cellular plasticity. At the molecular level, ZEB1 is linked in a double-negative feedback loop with the miR-200 family and miR-203, which are strong inducers of epithelial differentiation. Thus, aberrant ZEB1 expression stabilizes EMT and stemness, thereby promoting dissemination, metastasis, and drug resistance of cancer cells. We have validated the findings by showing that a depletion of ZEB1 in the KPC-mouse model of pancreatic cancer counteracts tumor cell plasticity and metastasis. Moreover, we detected that ZEB1 controls the Notch pathway and directly cooperates with the Hippo-pathway effector YAP in driving aggressive cancer types. We determined epigenetic modifications conferred by ZEB1, and screened for epigenetic drug to restore expression of its silenced target genes and to subsequently overcome therapy resistance. Despite their potent tumor-promoting effects, EMT-TFs are rarely mutated in cancer. This likely is due to the necessity for a transient expression and the associated plasticity of cancer cells, underscoring the important role of nonmutated genes in cancer progression.

Citation Format: Thomas Brabletz. Cellular plasticity in cancer: Driving force and therapeutic target [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr CN07-01.
Abstract LB-A11: Targeting DNA-PK and the DNA damage response via small molecule Ku inhibitors
Authors: Turchi John J. , Gavande Navnath , VanderVere-Carozza Pamela S. , Vernon Tyler , Pawelczak Katherine S.
Published: August 2018

The DNA dependent protein kinase (DNA-PK) is a validated target for cancer therapeutics that drives the DNA damage response (DDR) and plays a critical role as a primary sensor in the non-homologous end joining (NHEJ) DNA double strand break (DSB) repair pathway. Various anti-cancer therapeutic strategies mediate their cytotoxic effects by inducing DSBs, including ionizing radiation (IR), and clinical outcomes are directly related to the repair of DNA damage. Modulating the pathway responsible for repairing DSBs will have a profound impact on the efficacy of DNA damaging agents in the clinic. To date, development of inhibitors for DNA-PK has focused on targeting the active site with ATP mimetics. We have taken the novel and innovative approach to inhibiting DNA-PK via blocking the Ku 70/80 heterodimer interaction with DNA, a necessary step in DNA-PK activation. Exploiting this unique mechanism of kinase activation, we have identified a series of highly potent and specific DNA-PK inhibitors that impart their inhibitory activity via disruption of the binding of Ku protein to DNA ends. This novel approach affords significant advantages to current approaches in kinase inhibition. Novel derivatives of our initial hit inhibit DNA-PK catalytic activity at nanomolar concentrations and potentiate cellular sensitivity to DSB-inducing agents like etoposide and bleomycin. Data demonstrate that the cellular effects observed are a function of Ku inhibition and that this novel class of DNA-PK inhibitors can be further developed as anti-cancer therapeutics that can be used as an adjuvant to, or concomitant with radiotherapy and other cancer therapies that induce DNA damage.

Citation Format: John J. Turchi, Navnath Gavande, Pamela S. VanderVere-Carozza, Tyler Vernon, Katherine S. Pawelczak. Targeting DNA-PK and the DNA damage response via small molecule Ku inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A11.
Abstract LB-B10: Transcriptional landscape in the human Knock-in isogenic line for p53 polymorphism codon 72
Authors: Lee Seo-Young , Chung Sun-Ku , Park Jung-Hyun , Oh Soo A , Won Kyoung-Jae
Published: August 2018

Background: Codon 72, the most representative polymorphism of p53, commonly encodes the two kinds of amino acids, proline (P) or arginine (R), which is interestingly located within proline-rich domain to regulate p53-dependent transactivation. Although the previous reports have compared the polymorphism-mediated p53 transactivation, the results have some limitations that have been mined artificially on abnormal cells or plasmid-based expression. Thus, overcoming the concerns, we here established the isogenic lines to identify codon 72 polymorphic-dependent p53 transactivation.

Experimental design: We established isogenic line based on human induced pluripotent stem cells (hiPSC) with either the proline or arginine at codon 72 of p53 (P and R-type p53), using BAC-mediated homologous recombination system. In addition, to set the baseline mRNA expression level of p53 target genes, p53-null hiPSC has been generated with simultaneous reprogramming-CRISPR system. We applied RNA-sequencing technology to profile transcription patterns between three cell lines, p53-P, p53-R, and p53-null hiPSCs, under genotoxic stress, Doxorubicin treatment.

Results: Molecular signature analysis shows the significant difference of transcriptional patterns in PI3K-Akt signaling pathway, focal adhesion, cell-cell adhesion and Wnt signaling pathway by KEGG pathway mapping between P- and R-type. We identified both PI3K-Akt signaling related genes and focal adhesion related genes increased in P-type p53 than R-type p53, but expression patterns are similar with p53-null cell in focal adhesion. On the other hand, cell-cell adhesion related gene patterns down regulated comparing with p53-null cell. Lastly, R-type p53 gene expression shows the most increased pattern than other two cell types.

Conclusions: The isogenic line-based p53 polymorphism study reveals the differences intrinsically in the p53 polymorphism dependent-cell survival signaling, PI3K-Akt signaling pathway.

Acknowledgements: This work was supported by Korea Institute of Oriental Medicine Grant K17131 and National Research Foundation of Korea Grants 2013M3A9B4076487.

Citation Format: Seo-Young Lee, Sun-Ku Chung, Jung-Hyun Park, Soo A Oh, Kyoung-Jae Won. Transcriptional landscape in the human Knock-in isogenic line for p53 polymorphism codon 72 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B10.
Abstract LB-B23: Prodrug activation by designer enzyme targeted to CD123-expressing tumors
Authors: Mannervik Bengt , Sjödin Birgitta I.
Published: August 2018

We propose the combination of tumor-targeting antibodies with a drug-activating enzyme and a prodrug, which could be expected to give synergy and enhanced therapeutic efficacy in the treatment of leukemia and other malignancies. We have engineered human glutathione transferase (GST) enzymes for increased catalytic activity with a prodrug with the aim of delivering the enzyme selectively to a tumor. Antibodies, or similar specific binding proteins, will bind to selected epitopes of the cancer cells where the accompanying enzyme will exert its drug-activating function. The approach is generic and could be implemented with alternative binding proteins and GSTs with different functionalities. Myeloid neoplasms represent 20% of leukemias in children and the majority of the pediatric patients have acute myeloid leukemia (AML). There is a need for improved therapies to eradicate tumor cells with particular focus on leukemic stem cells (LSC), which are responsible for recurrent disease. We are designing, by protein engineering and directed in-vitro evolution, glutathione transferase (GST) enzymes that trigger cytotoxicity via chemically activating chemotherapeutic prodrugs. By fusing the enzyme with an antibody with affinity for epitopes selectively expressed by tumor cells, the release of the toxic agent will be targeted to the neoplastic tissue and minimize collateral damage to normal cells. For proof-of-concept a glutathione transferase enzyme activating the prodrug azathioprine is targeted via an anti-CD123 antibody to acute myeloid leukemia (AML) cells ex vivo. The target receptor CD123 is also expressed on LSC, but not on normal hematopoietic stem cells. Following binding to tumor cells GST will catalyze the conversion of the subsequently administered azathioprine to the anti-metabolite 6-mercaptopurine, which will interfere with nucleotide metabolism and DNA synthesis. Alternative prodrugs activated by other GSTs are also available, indicating the versatility of our approach. An engineered GST with 175-fold enhanced activity with azathioprine has been fused with an scFv-anti-CD123 antibody and the designer enzyme produced by heterologous expression. The recombinant fusion protein is catalytically fully competent and maintains the binding activity of the parental scFv antibody.

Citation Format: Bengt Mannervik, Birgitta I. Sjödin. Prodrug activation by designer enzyme targeted to CD123-expressing tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B23.
Abstract LB-B31: FGFR inhibition in endometrial cancer induces caspase-independent cell death that can be augmented with ABT-737
Authors: Packer Leisl , Byron Sara , Stehbens Samantha , Bonazzi Vanessa , Loch David , Wortmann Andreas , Gartside Mike , Waterhouse Nigel , Gunter Jennifer , Pollock Pamela M.
Published: August 2018

Endometrial cancer (EC) is the most commonly diagnosed malignancy of the female reproductive tract. Unfortunately, 15-20% of women demonstrate persistent or recurrent tumors that are refractory to current chemotherapies with an associated poor prognosis. Our laboratory identified activating mutations in Fibroblast Growth Factor Receptor 2 (FGFR2) in 12% (stage I/II) to 17% (stage III/IV) endometrioid endometrial tumors and have since shown in a large (n=970) multi-institutional cohort they are associated with shorter progression free and cancer specific survival. Although FGFR inhibitors are in clinical trials in several cancer types, no detailed study of the mechanism of cell death has been published. We now show that treatment with BGJ398, AZD4547 and PD173074 leads to the induction of mitochondrial depolarization and changes in metabolic flux in two endometrial cancer cell lines (JHUEM2 and AN3CA) carrying activating mutations (C383R and N550K respectively). Despite this mitochondrial dysfunction, we have convincingly shown that the cell death following FGFR inhibition was caspase-independent, as evidenced by the lack of caspase-3, -7, and -9 activation, absence of PARP cleavage, and the inability of the broad-spectrum caspase inhibitor, Z-VAD-FMK, to prevent cell death. Knockdown of EndoG and AIF, common mediators of caspase-independent death, had no effect. Detailed quantification of LC3 positive puncta shows an increase in autophagy in JHUEM2 and AN3CA cells treated with all FGFR inhibitors. Knockdown of ATG3, ATG7 and ATG12 resulted in a slight increase in Annexin positive cell death indicating that the autophagy was cytoprotective in this context. We have now confirmed this novel caspase-independent cell death is mitochondrial dependent as it can be blocked by overexpression of Bcl-2 and/or Bcl-XL. Importantly we have shown that the combination of FGFR inhibitors with the BH3 mimetic ABT737 can markedly augment this caspase-independent cell death which may have implications for the design of more effective clinical trials.

Citation Format: Leisl Packer, Sara Byron, Samantha Stehbens, Vanessa Bonazzi, David Loch, Andreas Wortmann, Mike Gartside, Nigel Waterhouse, Jennifer Gunter, Pamela M. Pollock. FGFR inhibition in endometrial cancer induces caspase-independent cell death that can be augmented with ABT-737 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B31.
Abstract 1037: Comprehensive molecular profiles of gastric cancer patient derived xenograft (PDX) models and its implication in precision cancer medicine
Authors: Lee Jae Eun , Choi Yoon Young , Lim Ju Yeon , Shin Su-Jin , Lee Gunho , Kim Eun Young , Son Taeil , Kim Hyoung-Il , Hyung Woo Jin , Noh Sung Hoon , Kim Hyunki , Jung Minkyu , Kim Sangwoo , Paik Soonmyung , Cheong Jae-Ho
Published: August 2018

Background: Recent cancer research is focused on precision medicine with the advent of next generation sequencing (NGS) technology and patient tumor derived model systems. Here, we present molecular characteristics of gastric cancer patient derived xenograft (PDX) models and explore the potential of molecularly defined PDX model based drug development.

Materials and Methods: We generated PDX models from patient tumors with advanced gastric cancer. The genomic alterations of tumors were profiled by whole exome sequencing (WES), RNA sequencing (RNAseq), targeted sequencing, in-situ hybridization (ISH) and immunohistochemistry (IHC). Further, we developed overcoming strategy of chemotherapy resistance mechanism by combination of signaling pathway inhibitor and standard chemotherapy regimen in “N-of-1” PDX trial.

Results: Thirty-five PDX models were successfully established and categorized into four subgroups of The Cancer Genome Atlas (TCGA) and Asian Cancer Research Group (ACRG) classifications: EBV (2.9%), MSI (20.6%), GS (23.5%) and CIN (52.9%) subtypes by TCGA while MSI type (11.4%), MSS/EMT type (17.1%), MSS/TP53+ (31.4%) and MSS/TP53- type (40.0%) by ACRG. In the protein levels by IHC, there were 21 cases (60.0%) of any RTK proteins overexpression; nine of HER2 (25.7%), 14 of EGFR (40.0%), and 16 of c-MET (45.7%). Five tumors (14.3%) were related to PTEN loss and 22 tumors (62.9%) showed p53 overexpression or null. Targeted sequencing identified that ERBB2 (25.7%), KRAS (11.4%), and CCND1 (11.4%) were found frequently amplified gene while PIK3CA (11.4%) and CTNNB1 (8.6%) were found most mutated genes. N-of-1 PDX trial demonstrated that the response to FOLFOX in PDX tumor was concordant with that of corresponding patient. In FOLFOX resistant tumors, multiple signaling pathways were up-regulated and inhibition of these signaling pathways was regressed tumor growth.

Conclusions: The utilization of molecularly catalogued gastric cancer PDX models will guide precision medicine for cancer therapy and be a useful tool for drug development and repurposing.

Citation Format: Jae Eun Lee, Yoon Young Choi, Ju Yeon Lim, Su-Jin Shin, Gunho Lee, Eun Young Kim, Taeil Son, Hyoung-Il Kim, Woo Jin Hyung, Sung Hoon Noh, Hyunki Kim, Minkyu Jung, Sangwoo Kim, Soonmyung Paik, Jae-Ho Cheong. Comprehensive molecular profiles of gastric cancer patient derived xenograft (PDX) models and its implication in precision cancer medicine [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 1037.
Abstract 1107: Cell-surface vimentin-positive macrophages like CTCs as novel biomarkers of metastatic gastrointestinal stromal tumors
Authors: Li Heming , Meng Qing H. , Noh Hyangsoon , Somaiah Neeta , Torres Keila E. , Xia Xueqing , Batth Izhar Singh , Joseph Cissimol P. , Mulder Zachary A. , Wang Ruoyu , Li Shulin
Published: August 2018

Background: Gastrointestinal stromal tumors (GISTs) are the most common gastrointestinal tract sarcomas and are heavily infiltrated with tumor-associated macrophages (TAMs). Biomarkers specifically capturing circulating tumor cells (CTCs) that can be used to noninvasively determine the disease status in GIST patients have been lacking.

Patients and Methods: To fill this gap, we captured both regular CTCs and TAMs from GIST patients' blood and tumor samples using our proprietary cell-surface vimentin (CSV) antibody 84-1 and the TAM markers CD14 and CD68 based on our previous established CTC detection method from 104 localized or metastatic sarcoma patients and 10 healthy donors. Also, freshly procured metastatic GIST tissues were obtained from four patients.

Results: We defined a rare population of CSV+ macrophage-like CTCs (ML-CTCs) in metastatic GIST patients' blood samples with expression of CD14 and CD68 but not CD45. Also, we demonstrated CSV+ ML-CTCs to be tumor microenvironment-derived. Metastatic GIST patients had markedly higher numbers of CSV+ ML-CTCs than localized GIST patients and healthy blood donors. However, regular CTC counts could not predict metastasis of GISTs but could for other types of sarcoma. Notably, CSV+ ML-CTCs were represented as M1-like macrophages in their phenotype and function.

Conclusions: CSV+ ML-CTCs are novel biomarkers for prediction of metastatic risk and therapeutic response in GIST patients. Routinely monitoring these cells will be an important approach to liquid biopsy analysis of GISTs.

Citation Format: Heming Li, Qing H. Meng, Hyangsoon Noh, Neeta Somaiah, Keila E. Torres, Xueqing Xia, Izhar Singh Batth, Cissimol P. Joseph, Zachary A. Mulder, Ruoyu Wang, Shulin Li. Cell-surface vimentin-positive macrophages like CTCs as novel biomarkers of metastatic gastrointestinal stromal 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 1107.
Abstract 1218: An eQTL and mQTL study of 147 genetic variants associated with prostate cancer susceptibility
Authors: Wang Xiaoyu , Cheng Anqi ,   the PRACTICAL Consortium, Stanford Janet L. , Dai James Y.
Published: August 2018

Genome-wide association studies by the PRACTICAL consortium have identified and validated 147 single nucleotide polymorphisms (SNPs) associated with prostate cancer (PCa) risk (Schumacher et al., Nat Genet 2017). However, most of these genetic variants are in noncoding regions, which poses a challenge for understanding the molecular mechanisms underlying these risk loci. Herein we study both expression quantitative trait loci (eQTL) and methylation quantitative trait loci (mQTL) among these PCa risk loci to identify molecular alterations associated with these genetic variants. Data were available for a Fred Hutchinson (FH)-based cohort of patients diagnosed with localized stage PCa, including SNP genotypes (Infinium iCOGS and OncoArray-500K BeadChips) from the PRACTICAL consortium and genome-wide DNA methylation (Infinium Human Methylation450 BeadChip) and gene expression (HumanHT-12 v4 BeadChip) profiling of primary tumor tissues obtained at radical prostatectomy. Missing genotypes were imputed using the October 2014 (Phase 3) release of the 1KGP data as the reference panel. These data were used to identify eQTL (N=355) and mQTL (N=377) in cis-acting associations where the transcripts or CpG sites are located within 2Mb (±1Mb) of the genetic variants. The Matrix eQTL software was used to conduct eQTL and mQTL analyses. In total, 4,300 SNP-transcript pairs and 104,600 SNP-CpG pairs were tested for eQTL and mQTL, respectively, among which we identified 64 eQTL-transcript pairs (37 PCa risk loci and transcripts in 54 genes) and 1,405 mQTL-CpG pairs (116 PCa risk loci and CpGs in 321 genes), after correction for multiple testing (FDR<0.05). The remaining 29 (20%) PCa risk loci were neither eQTLs nor mQTLs. Interestingly, 35 (95%) eQTLs were also mQTLs, while the majority of mQTLs were not eQTLs (Fisher's exact test of independence, p=0.005), suggesting that DNA methylation may have alternative biologic regulatory functions other than directly affecting gene expression. Several of these genes associated with eQTLs such as INCENP, NUDT11, RGS17, ASCL2, C10orf32, MSMB, BHLHA15, and IRX4 were previously found in PCa. Furthermore, we discovered a number of novel genes such as HLA-DRB3 and HLA-DRB4 associated with eQTLs in our data. Further validation using the TCGA-PRAD data is under way. In summary, we identified eQTLs and mQTLs among newly established genetic risk loci for PCa. These results provide new insight into both the genetic and epigenetic mechanisms through which these SNPs may alter susceptibility to PCa.

Citation Format: Xiaoyu Wang, Anqi Cheng, the PRACTICAL Consortium, Janet L. Stanford, James Y. Dai. An eQTL and mQTL study of 147 genetic variants associated with prostate cancer susceptibility [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 1218.
Abstract 122: Hypoxia-enhanced proliferation and effector function of NK cells
Authors: Moon Yunwon , Lim Seon Ah , Lee Kyung-Mi , Park Hyunsung
Published: August 2018

Hypoxia promotes tumor cell survival while providing immune cell shaping and resistance. Although NK cells function within hypoxic lymphatics and inflamed tissues, they were shown to mount impaired anti-tumor effector functions in ex-vivo hypoxic environment. This raises a question that additional cues exist in vivo to allow full NK cell activation. By varying the percentage and time of pO2 exposure ex-vivo, we were able to fine-tune a condition that reverses hypoxia-induced NK cell suppression to become highly proliferative and enhanced cytolytic phenotypes. We found that exposing NK cells to moderate hypoxia in the presence of IL-2 and feeder cells allowed HIF-1α stabilization and its target gene expression, metabolic changes toward glycolysis, specific upregulation of NKp44 receptor, and increased STAT3 phosphorylation with concomitant reduction of apoptosis and p16 senescence pathways. Therefore, the hypoxic exposure allows HIF-1α-mediated adaptation of mature NK cells toward highly proliferative and cytolytic NK phenotypes, which may occur in vivo at the time of tumor growth and inflammation. [This work was supported by the National Research Foundation of Korea (NRF), grants NRF-2013M3A9D3046248]

Citation Format: Yunwon Moon, Seon Ah Lim, Kyung-Mi Lee, Hyunsung Park. Hypoxia-enhanced proliferation and effector function of NK 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 122.
Abstract 1289: Crosstalk between methylation and alternative splicing in cancer
Authors: Kim Dongwook , Han Seonggyun , Lee Younghee
Published: August 2018

Background: DNA methylation in promoter regions leads to transcriptional silencing, such as of tumor suppressor genes in cancer. However, the functions of DNA methylation in the gene body (intragenic, i.e., exons and introns) have not yet been completely elucidated. Recently, hyper-methylation has been implicated in enhancing exon recognition by recruiting methyl-CpG-binding protein (MECP2) to hyper-methylated sites. In this study, we examined intragenic methylation in splicing regulatory elements (SREs: exonic splicing enhancer (ESE), exon splicing silencer (ESS), intronic splicing enhancer (ISE)) as a mechanism to understand how epigenetic factors contribute to alternative splicing events in cancer.

Hypothesis: Methylation patterns of SREs are involved in directing alternative splicing events in cancer.

Methods: We developed a splicing decision model to identify actionable methylation loci potentially affecting splicing events (i.e., exon skipping) among CpG sites in putative intragenic SRE regions. We used DNA methylation and RNA-Seq data for breast and lung cancer samples and normal cases from The Cancer Genome Atlas (TCGA). We additionally used hyper- and hypo-methylation data published in Selamat et al. (2013) as an independent lung cancer dataset. To identify regions hyper- and hypo-methylated in cancer, we performed an unpaired t-test for normally distributed data and a Mann-Whitney-Wilcox test for non-normally distributed data for differential methylation status (beta value) between cancer and normal cases. Multiple testing was accounted for by controlling the false discovery rate (p< 0.05 with FDR< 0.1).

Results: We investigated whether specific patterns of methylation were enriched in SRE regions affecting exon skipping. We found that for breast cancer samples, differential methylation status was enriched in SRE regions but not in non-SRE regions. Particularly, hypo-methylation in cancer showed a greater enrichment in ESE regions with putative exon skipping events. Similar results were observed in the both the TCGA lung cancer cases and an independent lung cancer dataset. We calculated GC contents and the methylation frequencies at each position of the hexameric sequence of SREs and further investigated whether certain SREs are prevalent to be with methylation or not. These data suggest that hypo-methylation in SRE regions may cause failure to recruit MECP2, therefore leading to exon skipping.

Conclusion: Our study suggests that intragenic methylation status is important for splicing regulation and may improve our understanding of how intragenic epigenetic markers play a role in gene regulation by affecting alternative splicing.

Citation Format: Dongwook Kim, Seonggyun Han, Younghee Lee. Crosstalk between methylation and alternative splicing in cancer [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 1289.
Abstract 1344: Autophagy contributes to therapeutic resistance in head and neck cancer
Authors: Eckers Jaimee , Skiba Justin , Senthilkumar Gopika , Nickel Kwang P. , Swick Adam D. , Kimple Randall J.
Published: August 2018

Background: In the curative setting for head and neck cancer (HNC) a common treatment is radiation combined with cetuximab, an antibody therapeutic targeting EGFR. Despite decades of research into improved treatments, therapeutic resistance remains a major challenge for this malignancy, with roughly 40% of patients developing recurrent disease. Recent evidence has suggested that autophagy, a cellular stress response, may be an additional contributor to therapy resistance, by protecting HNC cells from the cytotoxic effects of radiotherapy and the growth inhibitory effects of anti-EGFR treatment. The mechanism of radiation-induced autophagy is under current investigation.

Methods: Cell lines were source from commercial sources, cultured under recommended conditions, and identity confirmed by short tandem repeat testing. Induction of autophagy was detected by immunoblot flux assays for LC3 and p62, immunofluorescent staining of autophagic vesicles, LC3 reporter flux assay, and flow cytometry using acridine orange. The effect of autophagy inhibition was tested using clonogenic survival assays. Induction of apoptosis was analyzed by immunoblot against cleaved caspase and PARP and via AnnexinV staining.

Results: We evaluated a panel of both human papillomavirus (HPV) positive and negative HNC cell lines for autophagic response to both cetuximab (CTX) treatment and ionizing radiation (XRT). Flux assays revealed that both CTX and XRT treatment induced autophagy in a time- and dose-dependent manner. Immunofluorescent staining of LC3 to identify autophagic vesicles showed that a relatively small fraction of the total cell population is able to induce this response. Flow cytometry analysis demonstrated that autophagic cells were largely non-apoptoic. For example, in the UM-SCC47 cell line treated with CTX for 48 h, flow cytometry for autophagy (20.8%), apoptosis (13.7%) or dual staining (5.4%) suggests a cytoprotective role for autophagy. The addition of the ULK1 inhibitor, SBI-0206965) to CTX and XRT induced apoptosis as shown by caspase activity and AnnexinV staining and reduced clonogenic cell survival.

Conclusions: These preclinical studies have established the proof of concept for the cytoprotective effect of autophagy in response to anticancer treatments including EGFR inhibition and radiotherapy in HNC. Further, we have identified the addition of specific autophagy inhibitors to standard treatments as a potential strategy to overcome this mechanism of resistance.

Citation Format: Jaimee Eckers, Justin Skiba, Gopika Senthilkumar, Kwang P. Nickel, Adam D. Swick, Randall J. Kimple. Autophagy contributes to therapeutic resistance in head and neck cancer [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 1344.
Abstract 1699: Treatment with agonist anti-GITR antibody after chemotherapy enhances tumor immunity
Authors: Warner Allison Betof , Hirschhorn Daniel , Cohen Adam , Liu Cailian , Newman Walter , Sirard Cyndi , Wolchok Jedd , Merghoub Taha
Published: August 2018

Stimulation of glucocorticoid-induced tumor necrosis factor receptor (GITR) has been shown to enhance antitumor immunity by stimulating effector CD4+ and CD8+ T cells and attenuating suppression and depleting by CD4+Foxp3+ regulatory T cells (Treg). However, GITR monotherapy does not effectively control tumor growth. We hypothesize that cyclophosphamide (CTX), a cytotoxic chemotherapeutic agent with key immunomodulatory properties, can enhance the potency of GITR engagement anti-tumor effects. Further, we hypothesize that CTX can induce neoantigen formation by increasing tumor mutation burden. In this study, we administered high-dose CTX (250 mg/kg) followed the next day by an agonist anti-GITR antibody (DTA-1) to C56BL/6 mice bearing poorly immunogenic B16 melanoma tumors. We found that the combination therapy can cause regression of established tumors whereas the single agents did not. Mechanistically, we found that CTX administration results in tumor antigen release, which enhances the antitumor T cell response after treatment with DTA-1. This effect is lost when lower doses of CTX are given or CTX is administered prior to tumor inoculation, suggesting that tumor cell death and cross-presentation of tumor antigens to T cells is necessary. Furthermore, Treg cells are an important target of combination therapy. Intratumoral Treg levels were substantially reduced in treated mice, which greatly increased the ratio of T effector/Treg in the tumor. In conclusion, CTX and agonist anti-GITR antibody slows tumor progression and can induce regression of palpable, poorly immunogenic B16 melanoma tumors, likely by enhancing cross-priming and increasing the T effector/Treg ratio in the tumor. Thus, this combination warrants further investigation as a novel immunotherapy approach and provides rational for exploring combination of GITR agonist antibodies with chemotherapeutic treatments in clinical settings.

Citation Format: Allison Betof Warner, Daniel Hirschhorn, Adam Cohen, Cailian Liu, Walter Newman, Cyndi Sirard, Jedd Wolchok, Taha Merghoub. Treatment with agonist anti-GITR antibody after chemotherapy enhances tumor immunity [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 1699.
Abstract 1781: HPN536, a T cell-engaging, mesothelin/CD3-specific TriTAC for the treatment of solid tumors
Authors: Austin Richard , Aaron Wade , Baeuerle Patrick , Barath Manasi , Jones Adrie , Jones Susan D. , Law Che-Leung , Kwant Kathryn , Lemon Bryan , Muchnik Anna , Sexton Kenneth , Tatalick Laurie , Wesche Holger , Yu Timothy
Published: August 2018

Mesothelin (MSLN) is a GPI-linked tumor antigen overexpressed in a variety of solid tumors, including ovarian, pancreatic, lung and triple-negative breast cancer. Normal tissue expression is restricted to single-cell, mesothelial layers lining the pleural, pericardial, and peritoneal cavities. Overexpression of MSLN is associated with poor prognosis in lung adenocarcinoma and triple-negative breast cancer. MSLN has been used as cancer target antigen for numerous modalities, including immunotoxins, vaccines, antibody drug conjugates and CAR-T cells. Early signs of clinical efficacy have validated MSLN as target, but therapies with improved efficacy are still needed to address the significant, unmet medical need posed by MSLN-expressing cancers. HPN536 is a ~50-kDa antibody derivative called TriTAC (Tri-specific T cell Activating Construct) designed to simultaneously bind to MSLN on tumor cells and to CD3ε on T cells with an affinity of 1 nM and 14 nM, respectively. Transient bispecific binding leads to the formation of an immunological cytolytic synapse, T cell activation and redirected tumor cell killing. A third domain of HPN536 binds non-covalently to serum albumin with an affinity of 8 nM to extend serum half-life life. Because TriTACs are built using single domain antibodies, TriTACs are much smaller than full size antibodies and are anticipated to demonstrate improved penetration of human tumors compared to full sized antibodies. HPN536 is produced by eukaryotic cell culture and secreted as a highly stable, single polypeptide. It binds with similar affinity to human and cynomolgus MSLN, albumin and CD3. When incubated in co-cultures with resting, human or cynomolgus T cells and human tumor cells, T cells are induced to release cytokines, to proliferate, and to specifically lyse MSLN-positive target cells with EC50 values at single-digit picomolar concentrations. In an exploratory toxicological study in non-human primates, HPN536 was well tolerated and showed pharmacokinetics in support of weekly dosing in humans. Preclinical characterization suggests that HPN536 is an efficacious and safe novel therapeutic candidate for the convenient treatment of patients with MSLN-expressing malignancies.

Citation Format: Richard Austin, Wade Aaron, Patrick Baeuerle, Manasi Barath, Adrie Jones, Susan D. Jones, Che-Leung Law, Kathryn Kwant, Bryan Lemon, Anna Muchnik, Kenneth Sexton, Laurie Tatalick, Holger Wesche, Timothy Yu. HPN536, a T cell-engaging, mesothelin/CD3-specific TriTAC for the treatment of solid 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 1781.
Abstract 180: OPNa variant expression is associated with matrix mineralization in thyroid cancer cell lines
Authors: Ferreira Luciana B. , Lima Raquel T. , Bastos Ana Clara Santos da Fonseca , Silva Andreia Machado , Tavares Catarina , Pestana Ana , Rios Elisabete , Eloy Catarina , Sobrinho-Simões Manuel , Gimba Etel Rodrigues Pereira , Soares Paula
Published: August 2018

Osteopontin (OPN) and its three spliced variants (OPN-SV: OPNa, OPNb and OPNc) are overexpressed in several tumors and frequently associated with cancer progression. This holds true for papillary thyroid carcinoma (PTC), which is the most common variety of thyroid cancer (TC), being the histologic type which often presents desmoplasia (collagen deposition) and dystrophic calcification, including a fairly typical feature, the psammoma bodies (PB). This study aims to investigate the OPN-SV expression in classical variant of PTC (cPTC) and the role of OPN in calcification and collagen deposition into the extracellular matrix of thyroid cancer cell lines. Total OPN and OPN-SV expression was analyzed by immunohistochemistry and real time PCR in a series of 48 cPTC cases and three diffuse sclerosing PTCs. The association of OPN expression and the presence of PB as well as between PB in cPTC and the clinicopathological features of the tumors were evaluated. TPC-1 and c643 TC cell lines overexpressing OPN-SV were tested for the ability to promote calcification and to synthesize collagen in vitro. We also evaluated the expression of calcification markers, including osteocalcin and collagen I. Overexpression of OPNa transcripts was significantly associated with the presence of PB in cPTC samples. The presence of PB in cPTC was associated with younger patients and lymph node metastasis. Moreover, OPNa overexpression displayed a strong capacity to promote calcification and substantial collagen deposition in thyroid cancer cell lines. Upon OPN knockdown, the mRNA expression levels of calcification markers (osteocalcin and collagen I) were downregulated, further evidencing the involvement of OPN on activating these processes. Our data suggest that OPNa plays a role in the extracellular matrix calcification associated with cPTC. Basic research on the interactions between OPNa overexpression by tumor cells and the surrounding microenvironment can give clues for a better understanding of cPTC biology and phenotype.

Citation Format: Luciana B. Ferreira, Raquel T. Lima, Ana Clara Santos da Fonseca Bastos, Andreia Machado Silva, Catarina Tavares, Ana Pestana, Elisabete Rios, Catarina Eloy, Manuel Sobrinho-Simões, Etel Rodrigues Pereira Gimba, Paula Soares. OPNa variant expression is associated with matrix mineralization in thyroid cancer cell lines [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 180.
Abstract 1818: Activation of IGF-1R pathway and NPM-ALK G1269A mutation confer resistance to crizotinib in NPM-ALK positive lymphoma
Authors: Li Yanrong , Wang Kai , Zhang Jingdong
Published: August 2018

ALK-positive Anaplastic Large Cell Lymphoma (ALCL), harboring NPM-ALK fusion gene, is sensitive to dual ALK/MET inhibitor crizotinib. However, cancer cells eventually develop resistance to crizotinib after remarkable initial responses, the mechanisms of crizotinib resistance in NPM-ALK positive ALCL remained unclear.

To investigate the mechanisms of ALCL cells resistant to crizotinib, we established a model of acquired resistance to crizotinib by gradually exposing sensitive NPM-ALK-positive ALCL cell line to higher concentration of crizotinib. We found that NPM-ALK G1269A mutation were selected in 400nM resistant clones and activation of IGF-1R pathway. Then we generated a G1269A-mutated sub-lines by limiting dilution, which were sensitive to second-generation ALK inhibitor NVP-TAE684. IGF-1R inhibitor picropodophyllotoxin had no effect on these sub-clones and didn't change sensitivity to first and second generation ALK TKIs. But IGF-1R inhibitor increased sensitivity to crizotinib and NVP-TAE684 in 400nM crizotinib-resistant cells. Thus, we have shown that IGF-1R pathway and NPM-ALK G1269A mutation contribute to resistance to ALK TKIs and combined ALK/IGF1R inhibition improves therapeutic efficacy.

Citation Format: Yanrong Li, Kai Wang, Jingdong Zhang. Activation of IGF-1R pathway and NPM-ALK G1269A mutation confer resistance to crizotinib in NPM-ALK positive lymphoma [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 1818.
Abstract 2039: Cordycepin inhibits breast cancer migration and invasion by targeting epithelial to mesenchymal transition-inducing transcription factors (EMT-TFs)
Authors: Fu Junjiang , Wei Chunli , Cheng Jingliang , Khan Md. Asaduzzaman , Yang Lisha , Zou Hui
Published: August 2018

Cancer metastasis is one of the major causes of human death. The developmental stage epithelial to mesenchymal transition (EMT) is associated with cancer metastasis. Cordycepin (CD) is a natural compound, found in traditionally used medicinal mushroom Cordyceps. In this study, we have investigated the anticancer and antimetastatic activity of CD, and whether this compound regulates epithelial to mesenchymal transition-inducing transcription factors (EMT-TFs). By employing cell viability assay and real-time cell analysis in BT549, 4T1 and MDA-MB-435 cell lines, we found that CD can potentially inhibit cancer cell growth, migration and invasion. By using quantitative PCR (qPCR) and Western blot analysis, we also studied the expression of EMT associated proteins N-Cadherin and E-Cadherin, and their transcription factors TWIST1, SNAIL1, SLUG and ZEB1 in BT549 and 4T1 cell lines. By dual-luciferase reporter assay, we confirmed that CD is able to inactivate EMT signaling pathway with mediatory of TWIST1 and SLUG. CD treatment also inhibited the growth and metastasis of cancer cell-derived xenograft tumors in mouse model. Taken together, CD inhibits breast cancer cell migration and invasion by targets EMT-TFs, which might be a possible CD mechanism of antimetastatic action. In addition, CD derivatives by conjugating thymoquinone, a promising anti-cancer compound from nature, were designed to investigate whether there are dramatically improvements on their antimetastatic activities. In conclusion, this study suggests CD as a potential anticancer and antimetastatic molecule, which might be a promising therapeutic agent against breast cancer. Funding: This work was supported by the NSFC (81172049, 81672887).

Citation Format: Junjiang Fu, Chunli Wei, Jingliang Cheng, Md. Asaduzzaman Khan, Lisha Yang, Hui Zou. Cordycepin inhibits breast cancer migration and invasion by targeting epithelial to mesenchymal transition-inducing transcription factors (EMT-TFs) [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 2039.
Abstract 2098: Quantitative modeling as a systematic approach for drug combination evaluation in immuno-oncology (IO)
Authors: Helmlinger Gabriel , Kosinsky Yuri , Chu Lulu , Peskov Kirill , Voronova Veronika , Borodovsky Alexandra , Woessner Richard , Sachsenmeier Kris , Al-huniti Nidal
Published: August 2018

Objectives: Multiple strategies for eliciting and enhancing antitumor immunity are currently being evaluated. However, a more systematic approach is needed, to analyze and translate such results into clinic practice, while rationally designing combination therapies based on mechanistic understanding of potential synergistic effects (1). The objective of this study was to provide predictive simulations, via a quantitative systems pharmacology (QSP) model, capable of categorizing the types of synergistic effects that may arise from IO agent combinations, across realistic baseline conditions prevailing in the tumor microenvironment (TME).

Methods: The QSP model was developed and qualified using in vivo mouse data published in the literature and from internal research. The following pharmacologic modalities were calibrated: PD-L1/PD-1, CTLA-4, CXCR2, A2AR inhibition, and OX40 agonism. Various combination scenarios were simulated for these modalities, at four baseline conditions prevailing in different syngeneic murine models.

Results: Simulated efficacy results were highly dependent on the baseline conditions. Several combinations and monotherapies were effective only within a specific baseline TME phenotype. These findings were in agreement with experimental data (2). At baselines with higher levels of MDSC, best results were obtained for a PD-L1 mAb combined with either an OX40 agonist or a CXCR2 inhibitor, with 90% of complete responders. Anti (PD-L1 + CTLA-4) combinations showed high efficacy in Treg prevalence, but only moderate efficacy (22% complete responders), under baseline conditions of a dual (Treg + MDSC) immunosuppressive TME.

Conclusion: This work provides a quantitative modeling framework to comparatively predict responses to IO combinations, based on realistic baseline conditions prevailing in the TME, while revealing mechanistic interactions underlying such responses in IO combinations.

References:

1. Melero I et al. Nat Rev Cancer 2015;15:457-72.

2. Mosely S et al. Cancer Immunol Res 2016;5:29-41.

Citation Format: Gabriel Helmlinger, Yuri Kosinsky, Lulu Chu, Kirill Peskov, Veronika Voronova, Alexandra Borodovsky, Richard Woessner, Kris Sachsenmeier, Nidal Al-huniti. Quantitative modeling as a systematic approach for drug combination evaluation in immuno-oncology (IO) [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 2098.
Abstract 2195: Temporal and spatial dynamics of metastatic colonization revealed by 26 rapid-autopsy tumor biopsies from a TNBC patient
Authors: Huang Xiaomeng , Qiao Yi , Brady Samuel W. , Cohen Adam , Bild Andrea H. , Marth Gabor T.
Published: August 2018

Metastatic breast cancer is an advanced-stage disease in which the cancer cells have spread to distant organs. To understand the patterns of metastatic colonization in a patient who presented with aggressive disease, we have collected tumor biopsies at initial diagnosis, at mastectomy necessitated by the patient's relapse, twenty-six metastatic tumors across seven organs and two normal tissue control skin biopsies via a rapid autopsy procedure within hours after the patient's death.

All biopsy samples were subjected to 60X Illumina whole genome sequencing. Our analysis revealed extensive chromosomal changes including amplifications, deletions, LOH and translocations, as well as known driver mutations in RB1, TP53, and PTEN in all tumor samples. We used the CNV and LOH data to reconstruct the phylogenetic relationships among the tumor samples. We used the somatic SNV allele frequencies in copy number-normal regions to refine these phylogenetic relationships, and to establish the evolution of the tumor at the subclonal level across different metastatic sites, with an extension of our SubcloneSeeker algorithm.

Our analysis reveals the lung as the first metastatic site. In fact, this invasion occurred before relapse at the primary site in the breast, indicating early metastatic escape. This initial invasion was followed by distinct waves of massive metastatic expansion, beginning with the abdominal organs, then the lymph nodes, brain and bones. As the initial invasion, each metastatic wave involved other distinct sites in the lungs, these sites serving as incubators where subclones further evolved prior to seeding new metastatic sites. Finally, we see clear evidence for metastatic “recolonization” where such further evolved subclones invaded already established, earlier metastatic sites.

The high number of biopsied sites in this study, 30 in all, allowed us to reconstruct the evolution of the aggressive disease in our patient with unprecedented resolution, and to identify characteristic patterns of metastatic colonization. If confirmed in additional, similarly high resolution datasets, these patterns will lead to better understanding of the metastatic process, and guide effective clinical intervention.

Citation Format: Xiaomeng Huang, Yi Qiao, Samuel W. Brady, Adam Cohen, Andrea H. Bild, Gabor T. Marth. Temporal and spatial dynamics of metastatic colonization revealed by 26 rapid-autopsy tumor biopsies from a TNBC patient [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 2195.