bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4485: Enhancing antitumor immune responses with clinical BET bromodomain inhibitor RG6146
Authors: Hogg Simon J. , Wellinger Lisa , Rohle Daniel , Johnstone Ricky W.
Published: August 2019

The BET family of proteins bind to acetylated lysine residues on histone proteins and transcription factors to co-activate gene expression. BET proteins regulate the expression of oncogenes and can control the activity of various oncogenic transcription programs and have thereby emerged as therapeutic targets for the treatment of cancer. RG6146 is a novel non-covalent inhibitor of BET proteins that is in early phase clinical trials for the treatment of haematological and solid malignancies. The anti-tumor activity of BET inhibitors has primarily been attributed to tumor cell intrinsic effects, however increasing evidence suggests BET inhibitors modulate anti-tumor immune responses. Here, we examined the anti-solid tumor activity of RG6146 and evaluated the ability of RG6146 to enhance anti-tumor CD8+ T-cell responses.

To model anti-tumor CD8+ T-cell responses in vitro, syngeneic colon and breast tumor cells expressing ovalbumin (Ova) antigen were co-cultured with activated CD8+ T-cells derived from OT-1 transgenic mice. RG6146 functionally increased the activity of both wild-type and perforin-deficient OT-1 T-cells, leading to significantly enhanced T cell-mediated tumor cell death in a time- and dose-dependent manner. Mechanistic studies revealed that enhanced tumor cell death induced by RG6146 was dependent upon CD8+ T-cell derived tumor necrosis factor-α (TNF-α), independent of perforin/granzyme-dependent granule exocytosis. As RG6146 did not increase TNF-α production in CD8+ T-cells, we hypothesized RG6146 may sensitize tumors cells to TNF-α. Indeed, screening of cell lines revealed that BET inhibition significantly enhanced TNF-α-induced cell death in solid tumors of diverse origin. Underlying this response, we demonstrate using RNA- and ChIP-sequencing that BET inhibition suppresses transcription of pro-survival NF-kB target genes to elicit a potent pro-apoptotic phenotype. Finally, using syngeneic solid tumor models, we demonstrated that the adaptive immune system promotes the efficacy of RG6146 and evaluated the ability of RG6146 to therapeutically augment cancer immunotherapies in vivo.

Taken together, these data demonstrate that RG6146 is a potent BET bromodomain inhibitor with multi-faceted anti-cancer activity against solid tumors. We have identified a novel immunological TNF-α-dependent mechanism of bystander tumor killing by which BET inhibitors promote anti-tumor responses in vivo. Finally, we provide evidence that BET inhibition will augment the activity of cancer immunotherapies, establishing a strong rationale to evaluate these combinations in the clinic.

Citation Format: Simon J. Hogg, Lisa Wellinger, Daniel Rohle, Ricky W. Johnstone. Enhancing antitumor immune responses with clinical BET bromodomain inhibitor RG6146 [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 4485.
Abstract 4519: The reciprocal interaction between neutrophil extracellular traps and cancer cells impacts on their malignant potentials
Authors: Kajioka Hiroki , Kagawa Shunsuke , Ito Atene , Kuwada Kazuya , Kikuchi Satoru , Kuroda Shinji , Yoshida Ryuichi , Tazawa Hiroshi , Fujiwara Toshiyoshi
Published: August 2019

Although the neutrophil extracellular traps (NETs) are first recognized to function to trap invading microorganisms and eliminate them as a defense mechanism, the involvement of NETs in cancer metastasis is being elucidated. The mechanism of interaction between NET and cancer cells, however, remains to be fully understood. Here, we investigated the direct and indirect interaction between NETs and cancer. NETs were detected in surgically resected metastatic liver cancer tissue specimens. In direct co-culture, NETs actually attracted cancer cells with their web-like structure. Of note, indirect transwell co-culture of neutrophils and pancreatic cancer cells provoked NETs without the other stimuli. In addition, conditioned media derived from cancer cells also induced NETs. The co-culture of NETs and cancer cells dramatically accelerated their migration and invasion abilities. Further investigation revealed that NETs induced mesenchymal markers in cancer cells which was associated with upregulated epithelial to mesenchymal transition (EMT)-related transcriptional factors. Finally, the accelerated cancer migration and overexpression of mesenchymal markers induced via NETs were inhibited by pharmacological blockade of NETs including NADPH oxidase inhibitor, peptidylarginine deiminase inhibitor, and neutrophil elastase inhibitor. These results suggested that cancer cells and neutrophils reciprocally interact when they coexist and NETs drive malignant potentials via EMT, implicating NETs as a candidate therapeutic target.

Citation Format: Hiroki Kajioka, Shunsuke Kagawa, Atene Ito, Kazuya Kuwada, Satoru Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroshi Tazawa, Toshiyoshi Fujiwara. The reciprocal interaction between neutrophil extracellular traps and cancer cells impacts on their malignant potentials [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 4519.
Abstract 4540: Discover cell signaling pathways using antibody arrays
Authors: Tang Hao , Fang Jianmin , Kuang Zhizhou , Huang Ruo-Pan
Published: August 2019

Cell Signaling pathways are complex networks that are normally regulated and connected by multiple components. Analyzing signaling pathways is essential for understanding the molecular mechanism of human disease and drug development. However, it remains a great challenge to study pathways using traditional methods. Thus, a high-content and high-throughput method for simultaneous detection of multiple targets is necessary. Using a sandwich-based ELISA format, we have developed an antibody array technology which can simultaneously identify relative expression levels or phosphorylation levels of major signaling pathway proteins. This array-based system features a nitrocellulose membrane or glass slide solid support, each spotted with up to 71 different antibodies against a selected panel of classical signaling pathway proteins, including RTK, EGFR, MAPK, AKT, Apoptosis, TGFb, JAK/STAT, NFkB and Insulin Receptor pathways, spanning 182 total proteins. To test our system, we investigated the molecular mechanism of Camptothecin’s anti-cancer effect using several pathway arrays. Our array data suggest that Camptothecin-treatment induced DNA double-strand breaks in Jurkat cell activates the DNA damage pathways ATM and Chk2, and then further induces apoptosis through Caspase 3 and BAD. We also studied phorbol 12-myristate 13-acetate (PMA)’s effect on HeLa cells, and found that PMA induces the MAPK pathway through activation of Erk, MSK2, and RSK1. These array results are consistent with previous studies using traditional methods and were further confirmed by western blot analysis. Our studies demonstrate that pathway antibody arrays provide a rapid and efficient approach to study the mechanism of cell events, human diseases and drug effects. With this technology, researchers can quickly screen activation of multiple target proteins in one or multiple pathways in their experimental system.

Citation Format: Hao Tang, Jianmin Fang, Zhizhou Kuang, Ruo-Pan Huang. Discover cell signaling pathways using antibody arrays [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 4540.
Abstract 4544: PTP4A3 expression associated with increased invasion in esophageal adenocarcinoma
Authors: Wang Zhuwen
Published: August 2019

Esophageal adenocarcinoma (EAC) is the most rapidly growing solid malignancy with an overall 5-year survival rate of only 19%. The majority of EACs are diagnosed with advanced locoregional disease requiring neoadjuvant chemoradiation followed by esophagectomy. Understanding the molecular mechanisms associated with locoregional invasion are crucial for improving the treatment outcome.

Protein tyrosine phosphatase (PTP) type IVA member 3 (PTP4A3/PRL-3) is part of the PTP subfamily, which are phosphatases with dual-specificity. PTP4A3 has been shown to be overexpressed in a number of cancer types and often correlates with cell proliferation, migration, invasion, tumor growth, and metastasis. Here, we examine PTP4A3’s potential role in the invasive properties of EAC.

Methods: We analyzed PTP4A3 mRNA levels of 122 chemo-naïve EAC samples using Human Gene 2.1 ST Arrays and compared these to available histological features. We also used RNA-seq analysis to examine the expression of PTP4A3 in 68 samples that consisted of Barrett’s Esophagus, low-grade dysplasia, high-grade dysplasia, and EAC. PTP4A3 knockdown was achieved by using small interfering RNA and a rhodamine derivative (BR-1) inhibitor and was confirmed by both RT-PCR and Western blot. WST and clonogenic assays were performed to assess cell proliferation in cell lines derived from EAC tumors. Cell invasion and migration were measured by matrigel invasion and cell migration assays.

Results: PTP4A3 was significantly overexpressed in EAC samples compared to a cohort of normal esophageal samples (p<0.001). In addition, the expression of PTP4A3 increased with progression from Barrett’s to EAC. PTP4A3 was overexpressed in 15 EACs relative to 26 Barrett’s samples on U133A Array, which was confirmed in the 68-sample RNAseq progression cohort (p<0.01). Analysis of 122 EAC samples indicated that overexpression of PTP4A3 was significantly associated with decreased survival (p<0.05) and correlated with poor differentiation, high immune cell infiltration, and a high desmoplastic response (p<0.05). Inhibition of PTP4A3 in EAC cell lines led to a decrease in cell invasion and migration (p<0.01). However, there was no significant change in cell proliferation following knockdown.

Conclusion: PTP4A3 is frequently overexpressed in EAC and is associated with decreased survival. Inhibition of PTP4A3 decreased matrigel invasion and cancer cell migration, suggesting that PTP4A3 may play a role in the invasion and metastases of EAC cells and could serve as a biomarker of more aggressive disease.

Note: This abstract was not presented at the meeting.

Citation Format: Zhuwen Wang. PTP4A3 expression associated with increased invasion in esophageal 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 4544.
Abstract 4611: Identifying drivers of mammary tumorigenesis & elucidating the mechanisms of cancer initiation in DNA replication defective Chaos3 mice
Authors: Winters Marquita , Schimenti John
Published: August 2019

Distinguishing commonly deleted genes as drivers or passengers of human cancers are key to delineating mechanisms of tumor initiation. The “Chaos3” mouse model of spontaneous breast cancer carries a missense mutation in a gene called Mcm4 (minichromosome maintenance 4), causing destabilization of the MCM2-7 replicative helicase, which in turn causes elevated genomic instability. Approximately 80% of female mice homozygous for this mutation in the C3HeB/FeJ strain background develop mammary tumors with an average latency of 12 months. Genomic analysis of these tumors revealed a recurrent subset of genes with copy number alterations such as Arid1a and Nf1. This project aims to: 1) test whether the genes Arid1a and Nf1 act as mammary tumor drivers in the Chaos3 breast cancer model; 2) understand the genome-wide effects of Arid1a loss in mammary tumorigenesis; 3) identifying potential therapeutic targets for Arid1a-deficient mammary tumors; and 4) unravel the mechanism by which destabilization of MCM2-7 replicative helicase leads to cancer onset. To achieve these aims, several experiments such as conditional mutagenesis, CUTandRUN, RNA-seq, and replication timing profiling were conducted to address my aims. Preliminary data indicates that heterozygous loss of Arid1ain Chaos3 mammary tumors produces a distinct transcriptional profile compared to mammary tumors without Arid1a deletion. Additionally, Chaos3 primary cells have markedly altered replication timing patterns in certain regions of the genome that might explain increased susceptibility to mutations. My experiments can validate new candidates like Arid1a as a driver of sporadic breast cancer in humans. In addition, these studies will not only lend insight into how key tumor suppressors are deleted in the Chaos3 model, but also potentially reveal features of genomic regions that are particularly susceptible to genetic or environmental perturbations to DNA replication. The Chaos3 sporadic breast cancer mouse model mimics sporadic human luminal breast cancer and can be used as a tool to better understand the biology and genetics of a common disease that plagues women worldwide.

Citation Format: Marquita Winters, John Schimenti. Identifying drivers of mammary tumorigenesis & elucidating the mechanisms of cancer initiation in DNA replication defective Chaos3 mice [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 4611.
Abstract 4648: Cellular senescence and transformation induced by an oncogenic EML4-ALK fusion gene in normal and immortalized human cells
Authors: Miyanaga Akihiko , Horikawa Izumi , Matsumoto Masaru , Oike Takahiro , Beck Jessica , Tanaka Hiromi , Robles Ana I. , Seike Masahiro , Gemma Akihiko , Harris Curtis C.
Published: August 2019

Background: Chromosomal inversions involving Anaplastic lymphoma kinase (ALK) and Echinoderm Microtubule Associated Protein Like 4 (EML4) generate a fusion protein EML4-ALK with the constitutive ALK kinase activity in non-small cell lung carcinoma (NSCLC). The basic understanding of EML4-ALK remains insufficient due to the lack of functional studies using normal human cells.

Material and Method: We investigate the activities of EML4-ALK in mortal and immortalized normal human cells.

Results: The expression of EML4-ALK in normal, mortal human fibroblasts caused, through its ALK kinase activity, the early induction of cellular senescence with senescence-associated β-galactosidase activity, upregulation of p16INK4A and p21WAF1, telomere shortening and fusions, and accumulated DNA damage. In contrast, when EML4-ALK was expressed in telomerase reverse transcriptase (hTERT)-immortalized normal human fibroblasts, the cells showed accelerated proliferation and anchorage-independent growth, revealing its transformation activity. No chromosome aberration, no mutation or loss of p53, nor impairment of the p16INK4A response was associated with this transformation, likely reflecting clinical features of EML4-ALK-positive NSCLC. In both mortal and immortalized cells, EML4-ALK induced the phosphorylation of STAT3, which is involved in both cellular senescence and transformation. EML4-ALK was also able to transform hTERT-immortalized human bronchial epithelial cells, a cell type relevant to NSCLC.

Conclusions: Our data not only validate that EML4-ALK functions as an oncogene in human cells, but also suggest that telomerase-mediated immortalization manifests the oncogenic activity of EML4-ALK, switching from its senescence-inducing activity. This study also provides the isogenic pairs of human cell lines with and without EML4-ALK as an in vitro model for screening and testing candidate drugs.

Citation Format: Akihiko Miyanaga, Izumi Horikawa, Masaru Matsumoto, Takahiro Oike, Jessica Beck, Hiromi Tanaka, Ana I. Robles, Masahiro Seike, Akihiko Gemma, Curtis C. Harris. Cellular senescence and transformation induced by an oncogenic EML4-ALK fusion gene in normal and immortalized human 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 4648.
Abstract 4714: Preclinical evaluation of a novel LSD1 inhibitor SYHA1807 for the treatment of small cell lung cancer
Authors: Wu Lingyun , Yang Hanyu , Zhao Lele , Sun Jianjun , Liu Jian , Xia Yuanfeng , Chan Chichung , Chen Shuhui
Published: August 2019

Histone post translational modification is an important epigenetic regulation to activate or suppress specific gene expressions. Methylation which is dynamically regulated by methyltransferase and demethylase (e.g. lysine specific demethylase) is one of the mechanisms and is critical for cell cycle and differentiation. Lysine specific demethylase 1 (LSD1) is the first identified histone modifying enzyme that removes methyl from histone lysine and arginine residues. Its overexpression has been found in many human cancers, like blood, lung and breast cancers. Small cell lung cancer is a neuroendocrine cancer characterized as low differentiation, rapid metastatic spread and resistance to regular chemotherapy. Aberration of epigenetic regulation is a common feature in small cell lung cancer, like down regulation of tumor suppression gene and up regulation of oncogenes. LSD1 expression of small cell lung cancer patients is up regulated in tumor tissue but not in normal tissues. Dysregulated histone methylation may contribute to the tumor malignance and treatment resistance. Taken together, LSD1 activity inhibition may provide a promising treatment option for small cell lung cancer. A challenge in LSD1 drug development is how to maintain optimal therapeutic effects while reducing mechanism related hematological toxicity. Through scaffold hopping and thoroughly SAR exploration, a novel LSD1 inhibitor SYHA1807 has been identified. The exposure level of this candidate at lung tissue is about four times higher than in bone marrow. The ideal drug tissue distribution characteristics may provide further safety assurance in clinical application. Detailed ADME/PK, in vitro, in vivo properties and preliminary safety evaluation results will be presented.

Note: This abstract was not presented at the meeting.

Citation Format: Lingyun Wu, Hanyu Yang, Lele Zhao, Jianjun Sun, Jian Liu, Yuanfeng Xia, Chichung Chan, Shuhui Chen. Preclinical evaluation of a novel LSD1 inhibitor SYHA1807 for the treatment of small cell 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 4714.
Abstract 4718: SR-4370, a potent and selective inhibitor of class I HDACs, suppresses AR signaling and in vivo prostate tumor growth
Authors: Mahmud Iqbal , Tian Guimei , Wang Jia , Stowe Ryan , Huo Zhiguang , Zhang Yushan , Purayil Hamsa Thayek , Helm Eric , Drashansky Theodore , Avram Dorina , Daaka Yehia , Roush William R. , Liao Daiqing
Published: August 2019

Androgen receptor (AR) is an androgen-activated transcription factor and drives prostate cancer (PCa) progression. Class I HDACs 1-3 are critical for activating AR-mediated transcription. Thus, targeting these HDACs is a promising strategy for treating PCa. Notably, along with significant adverse effects, several FDA-approved HDAC inhibitors broadly inhibiting different HDACs were ineffective for treating castration-resistant prostate cancer in clinical trials. Significantly, entinostat, an aminobenzamide analog specific to HDACs 1-3, extended overall survival for patients with breast cancer resistant to endocrine therapy. These observations suggest that HDACi selective to HDACs 1-3 may be effective for treating solid tumors including PCa. We have recently discovered the novel benzoylhydrazide class of HDAC inhibitors highly specific to HDACs 1-3. An optimized analog, SR-4370, exhibited low µM to nM potency against HDACs 1-3. SR-4370 markedly suppressed AR signaling, PCa cell proliferation in vitro, and prostate tumor growth in vivo. Gene expression profiling experiments revealed that SR-4370 downregulated AR, AR-Vs and AR target genes as well as the MYC oncogenic network. Chromatin accessibility assay using ATAC-seq showed that SR-4370 altered chromatin states in PCa cells. The chromatins with AR-binding sites became inaccessible on SR-4730 treatment, indicating that altered chromatin accessibility may contribute to the inhibition of AR signaling. Interestingly, SR-4370 sensitized C4-2 cells to enzalutamide. In PCa xenograft models, SR-4370 was effective to suppress tumor growth in vivo. Importantly, SR-4370 was well tolerated and did not cause observable adverse effects as judged by body weight and blood chemistry tests of treated mice. Our data suggest that SR-4370 may be a safe and clinically applicable treatment for advanced PCa refractory to current frontline treatments. (Supported by UFHealth Cancer Center, Florida Breast Cancer Foundation, James and Esther King Biomedical Research Program, and Bankhead-Coley Cancer Research Program, Florida Department of Health)

Citation Format: Iqbal Mahmud, Guimei Tian, Jia Wang, Ryan Stowe, Zhiguang Huo, Yushan Zhang, Hamsa Thayek Purayil, Eric Helm, Theodore Drashansky, Dorina Avram, Yehia Daaka, William R. Roush, Daiqing Liao. SR-4370, a potent and selective inhibitor of class I HDACs, suppresses AR signaling and in vivo prostate tumor growth [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 4718.
Abstract 4728: IFNγ signaling drives EZH2 degradation to induce MHC-II expression in melanoma
Authors: James Jamaal L. , Opalenik Susan R. , Toren Abigail , Cook Rebecca S. , Balko Justin M.
Published: August 2019

Background: The molecular mechanisms governing anti-PD-1 resistance in melanoma are not fully understood. Suboptimal transcriptional and molecular response to interferon-gamma (IFNγ) is commonly identified in tumors with intrinsic or acquired resistance to anti-PD-1. Epigenetic regulators have been shown to reprogram IFNγ responses in some cases, though the mechanism is not clear. The ability of melanoma cells to express MHC-II is a positive clinical predictor to anti-PD-1 therapy, and MHC-II is induced only on cells with robust IFNγ responses. Enhancer of zeste homolog 2 (EZH2), an epigenetic regulator with both methyltransferase-dependent and -independent function, can suppress IFNγ target genes and is frequently overexpressed in melanoma. However, the mechanisms whereby EZH2 interacts with the IFNγ pathway and its role in melanoma-specific MHC-II expression is not known.

Methods: HLA-DR (MHC-II)-proficient (A375, SKMEL28 and SKMEL5) and -deficient (CHL-1 and MEWO) melanoma cell lines were stimulated with IFNγ for up to 48h, and the modulation of EZH2 protein and function were studied temporally. To test the role of EZH2 in MHC-II (HLA-DR) expression in IFNγ responses, genetic (siRNA) and chemical (GSK343) EZH2 inhibition was utilized.

Results: EZH2 was highly expressed in all cell lines tested, regardless of HLA-DR-proficiency. In 2 out of 3 HLA-DR-inducible melanoma cell lines, EZH2 protein expression was downregulated (4-24 hrs) in response to IFNγ stimulation, without effects on EZH2 mRNA expression or downstream tri-methylated H3K27 expression. Blocking proteosomal degradation with MG-132 reversed the IFNγ-induced decrease in EZH2 expression in HLA-DR-proficient cell lines. After siRNA knockdown of EZH2, SKMEL28 cells increased constitutive HLA-DR expression, while A375 cells became more sensitive to IFNγ-induced HLA-DR upregulation. In addition, IFNγ-induced HLA-DR was increased in A375 cells with EZH2 inhibition using GSK343.

Conclusion: This work demonstrates a potential role for EZH2 in suppressing IFNγ responses, including the induction of MHC-II, in melanoma cells. EZH2 is subject to proteosomal degradation in MHC-II-inducible cell lines, which, in part, contributes to the expression of MHC-II.

Citation Format: Jamaal L. James, Susan R. Opalenik, Abigail Toren, Rebecca S. Cook, Justin M. Balko. IFNγ signaling drives EZH2 degradation to induce MHC-II expression in melanoma [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 4728.
Abstract 4729: Targeting c-MYCdegradation as a novel therapeutic strategy for osteosarcoma: Studies in canine and human osteosarcoma cells
Authors: Yang Ya-Ting , Yuzbasiyan-Gurkan Vilma , Tepe Jetze J.
Published: August 2019

Osteosarcoma (OS) is the most common bone tumor in both humans and dogs, and has a nearly ten-fold higher incidence in dogs than humans. For the past twenty years, despite advances in treatment of other cancers, the overall survival rates for OS have stagnated. Thus, there is a great need for novel therapeutic strategies. In osteosarcoma, MYC activation is critical for both initiation and maintenance of tumorigenesis and tissue invasion. While targeting c-MYC is a rational strategy to treat OS, c-MYC has evaded therapeutic manipulation to date. In this study, we evaluated using a novel proteasome modulator, TCH-165, which induces c-MYC degradation, and compared it with convention treatments in in vitro studies. We demonstrated dose dependent decrease of c-MYC protein expression with TCH-165 treatment. Utilizing seven canine OS cell lines and three human OS cell line, we determined that the IC50 values for TCH-165 range from 2.2 to 11.6 μM in canine OS, 3.1 to 4.5 μM in human OS cell lines, while IC50 of several primary canine ranged from 22.3 μM to >200μM, suggesting a wide margin of safety. In cell cycle studies, we showed that TCH-165 resulted in G1 arrest after 12 hours of incubation. In addition, we examined the potential of combing TCH-165 with convention therapeutic agents. We selected carboplatin, one of the standard anti-tumor drugs for OS. Combination index calculations using TCH-165 and carboplatin in the canine D17 cell line support synergistic effects of this combination. The data point to potential novel avenues for treatment of OS which can be further examined in proof of concept studies in dogs with OS, serving as a relevant translational model, and accelerate drug development for human OS patients.

Citation Format: Ya-Ting Yang, Vilma Yuzbasiyan-Gurkan, Jetze J. Tepe. Targeting c-MYCdegradation as a novel therapeutic strategy for osteosarcoma: Studies in canine and human osteosarcoma 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 4729.
Abstract 4741: Re-evaluating the role of P-glycoprotein in the resistance of high-risk neuroblastoma to standard-of-care chemotherapies
Authors: Atkinson Caroline , Tactacan Carole M. , Kamili Alvin , Saletta Federica , Gana Christine , Eden Georgina L. , Mayoh Chelsea , Lock Richard B. , Norris Murray D. , Haber Michelle , Gifford Andrew J. , Trahair Toby N. , Fletcher Jamie I.
Published: August 2019

Background: Neuroblastoma is the most common extracranial solid malignancy in children, comprising 15% of cancer related deaths. Despite intensive treatment, patients with high-risk neuroblastoma (HR-NB) have a survival rate of ~50%, largely due to intrinsic or acquired drug resistance. The multidrug transporter P-glycoprotein (P-gp; ABCB1) effluxes several conventional agents used in HR-NB induction therapy, including doxorubicin, vincristine and etoposide, as well as the ALK inhibitor crizotinib. We observed high P-gp expression in HR-NB cell lines and patient-derived xenograft (PDX) models, so sought to assess the prevalence of P-gp expression and its role in resistance to standard-of-care chemotherapies and relevant targeted agents.

Methods and Results: Using RNA-sequencing, immunohistochemistry and western blot on panels of neuroblastoma tumour samples, PDX models and cell lines, we demonstrated that high ABCB1/P-gp expression is frequent in HR-NB. Analysis of ABCB1 levels in large patient tumor datasets suggests that high expression is attributable to the sympathoadrenal lineage of the disease, that high expression is more common in HR-NB than in most other cancers, and that high relative expression of ABCB1 in HR-NB tumours is associated with poorer outcome, consistent with its multidrug transporter function. We demonstrated that the P-gp inhibitor tariquidar and P-gp knockdown (shRNA) both strongly sensitize cultured high P-gp expressing neuroblastoma cells to vincristine, doxorubicin and etoposide but not to ALK inhibitors. Further, P-gp knockdown sensitized human neuroblastoma xenografts to vincristine, substantially extending survival.

Conclusions: Elevated P-gp expression is common in HR-NB and can be sufficient to confer resistance to standard-of-care chemotherapies in model systems. Our findings suggest that tumour P-gp levels might be used to guide treatment options for individual patients, and to avoid ineffective treatments. The potential of P-gp inhibitors as adjuncts to conventional chemotherapy for HR-NB should be further investigated.

Citation Format: Caroline Atkinson, Carole M. Tactacan, Alvin Kamili, Federica Saletta, Christine Gana, Georgina L. Eden, Chelsea Mayoh, Richard B. Lock, Murray D. Norris, Michelle Haber, Andrew J. Gifford, Toby N. Trahair, Jamie I. Fletcher. Re-evaluating the role of P-glycoprotein in the resistance of high-risk neuroblastoma to standard-of-care chemotherapies [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 4741.
Abstract 4743: Using genome-wide CRISPR screen to understand resistance mechanisms to PCA062, a P-cadherin targeting antibody-drug conjugate
Authors: Tam Angela , Zambrowski Mark , Seiss Katherine , Liu Si-Qi , Abrams Tinya , Caponigro Giordano , Tschantz William , Campbell Jennifer , DAlessio Tony , Sheng Qing
Published: August 2019

P-cadherin (PCAD) is a member of the cadherin family that mediates calcium dependent cell-cell contacts in adherens-type junctions of epithelium. Expression of P-cadherin is high in malignant tumors of epithelial origin, such as breast, esophagus, head and neck cancers, but low in normal tissues. This expression pattern makes P-cadherin a potential good target for antibody-drug conjugates (ADCs). PCA062 is a first-in-class antibody drug conjugate targeting P-cadherin. PCA062 consists of a fully human anti-P-cadherin antibody of the IgG1/κ subtype, a non-cleavable bi-functional linker (SMCC) and a maytansine-derived cytotoxic payload (DM1, with a target average drug to antibody ratio (DAR) of 3.8).

PCA062 activity was examined in a collection of cell lines expressing high level of PCAD. A subset of these PCAD high cell lines are resistant to PCA062 treatment while they remain sensitive to DM1, suggesting defects in the process of PCA062 uptake or the processing and release of DM1 into the cytoplasmic compartment. PCA062 internalization rate was measured by the uptake of a fluorescent dye labeled anti-PCAD antibody (CQY684, the Ab portion of PCA062) in both PCA062 sensitive and resistant lines. PCA062 resistant lines show slower CQY684 internalization as compared to PCA062 sensitive lines, indicating a defect in ADC internalization may contribute to PCA062 resistance. To explore additional resistance mechanisms to PCA062 as well as to find critical components for PCA062 internalization, a genome wide CRISPR screen was performed in PCA062 sensitive HCC1954 and in PCA062 resistant KYSE510 cell line in the presence and absence of PCA062 and DM1 to look for genes that when knocked out may specifically modulate PCA062 sensitivity. The multi-drug resistant gene MRP1 is a strong hit for PCA062 sensitization in both PCA062 resistant KYSE510 and PCA062 sensitive HCC1954 cells. Lysosomal transporter SLC46A3 and Saga transcription complex components are strong rescue hits for PCA062 in HCC1954. These data suggest that in addition to target expression level and cell intrinsic sensitivity to payload, genes involved in ADC internalization and payload cytoplasmic accumulation will also impact tumor cell sensitivity to ADCs.

Citation Format: Angela Tam, Mark Zambrowski, Katherine Seiss, Si-Qi Liu, Tinya Abrams, Giordano Caponigro, William Tschantz, Jennifer Campbell, Tony DAlessio, Qing Sheng. Using genome-wide CRISPR screen to understand resistance mechanisms to PCA062, a P-cadherin targeting antibody-drug conjugate [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 4743.
Abstract 4748: RICTOR signaling modulates the activity of EGFR tyrosine kinase inhibitors (TKI) in EGFR-mutated non-small cell lung cancer (NSCLC)
Authors: Fan Ni , Zou Yiyu , Halmos Balazs , Perez-Soler Roman , Cheng Haiying
Published: August 2019

Background: Molecularly-targeted therapy has significantly improved the therapeutic efficiency and clinical outcome in a subgroup of NSCLC patients. However, drug resistance inevitably emerges, such as resistance to EGFR TKIs in EGFR-mutated NSCLC. RICTOR is a key component of the mTORC2 complex and plays important roles in cell survival, proliferation, migration and invasion. RICTOR has also been suggested to play a part in the regulation of drug resistance to HER2-targeted therapy in HER2-amplified breast cancer, a niche close to EGFR-targeted therapy. In our current study, we hypothesize that RICTOR modulates the activity of EGFR TKIs in EGFR-mutated NSCLC. Co-targeting RICTOR and EGFR signaling pathways could be a new therapeutic strategy for patients with EGFR-mutated lung cancer.

Results: Genetically modified EGFR-mutated NSCLC cell lines with different RICTOR expression levels have been developed. RICTOR knockdown significantly increases the sensitivity to all three generations of TKI inhibitors (erlotinib, afatinib and osimertinib), whereas overexpression of RICTOR renders the cells more resistant to EGFR TKIs. In addition, RICTOR knockdown restores the sensitivity of erlotinib-resistant HCC827 cells due to AXL overexpression (IC50: 216µM vs. 0.256 µM with RICTOR knockdown, P<0.001). Pharmacological inhibition of the RICTOR signaling pathway with the mTOR1/2 inhibitor (sapanisertib) also improves EGFR TKI sensitivity. Taken together, RICTOR signaling modulates the efficacy of EGFR TKIs in the EGFR-mutated lung cancer models.

Conclusion: Our study provides a new insight into the role of RICTOR signaling modulation in increasing efficacy of EGFR TKI therapy in EGFR-mutated NSCLC. The combination of mTOR1/2 inhibitor and EGFR inhibitor could be a potential novel strategy leading to better therapeutic efficacy.

Note: This abstract was not presented at the meeting.

Citation Format: Ni Fan, Yiyu Zou, Balazs Halmos, Roman Perez-Soler, Haiying Cheng. RICTOR signaling modulates the activity of EGFR tyrosine kinase inhibitors (TKI) in EGFR-mutated non-small cell lung cancer (NSCLC) [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 4748.
Abstract 4749: Acquired resistance to BET-PROTACs caused by genomic alterations in core components of E3 ligase complexes
Authors: Riley-Gillis Bridget , Zhang Lu , Vijay Priyanka , Shen Yu
Published: August 2019

Proteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that hijack endogenous E3 ubiquitin ligases to induce ubiquitination and subsequent degradation of protein of interest. Recently, it has been shown that PROTACs with robust in vitro and in vivo activities and, in some cases, drug-like pharmaceutical properties can be generated using small molecule ligands for the E3 ligases VHL and CRBN. These findings stoked tremendous enthusiasm on using PROTACs for therapeutics development. Innate and acquired drug resistance often underlies therapeutic failures, particularly for cancer therapy. With the PROTAC technology progressing rapidly towards therapeutic applications, it would be important to understand whether and how resistance to these novel agents may emerge. Using BET-PROTACs as a model system, we demonstrate that resistance to both VHL- and CRBN-based PROTACs can occur in cancer cells following chronic treatment. However, unlike what was often observed for many targeted therapeutics, resistance to BET-PROTACs did not result from secondary mutations that affect compound binding to the target. In contrast, acquired resistance to both VHL- and CRBN-based BET-PROTACs was primarily caused by genomic alterations that compromise core components of the relevant E3 ligase complexes.

Citation Format: Bridget Riley-Gillis, Lu Zhang, Priyanka Vijay, Yu Shen. Acquired resistance to BET-PROTACs caused by genomic alterations in core components of E3 ligase complexes [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 4749.
Abstract 4751: Overcoming cisplatin resistance of esophageal squamous cell carcinoma by targeting RAC1-regulated Warburg effect
Authors: Zeng Ruijie , Zheng Chunwen , Gu Jinge , Peng Liu , Zhang Haixia , Feng Pan , Chen Yang , Xu Xiue , Xu Liyan , Li Enmin
Published: August 2019

Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy with poor prognosis. Despite treatment advances, development of chemo-resistance remains a major challenge in treating ESCC patients. Ras-related C3 botulinum toxin substrate 1 (RAC1) is essential in regulating cancer progression. However, its role in chemo-resistance of ESCC and the underlying mechanisms are still unknown. In this study, we found that higher levels of RAC1 expression were associated with poorer disease-free survival (p=0.014) and overall survival (p=0.013) by immunohistochemistry in human ESCC samples (n=106). The over-expression of RAC1 was correlated with enhanced cell proliferation, migration, invasion and chemo-resistance in both KYSE150 and KYSE510 cells, while the knockdown of RAC1 produced the opposite results. The combination of RAC1 inhibitor EHop-016 with cisplatin significantly promoted cell viability reduction, cycle arrest at G2/M phase and apoptosis when compared with the single treatment. To examine the in vivo effects of RAC1 inhibition on chemo-resistance, we performed xenograft implantation studies in immunocompromised mice (n=10 per group). In the combination group, the tumor volume was significantly reduced to 21.4% (p<0.001) as compared to the single treatment groups (cisplatin: 62.7%, RAC1 inhibitor: 58.5%). Mechanistically, the RNA-Seq data indicated that the glycolytic pathway was significantly down-regulated in the EHop-016-treated group (p<0.001, Log2FC=1.86) and the combination group (p<0.001, Log2FC=1.76), as compared to the control group. Moreover, the qRT-PCR and western blot results confirmed that RAC1 inhibitor alone or the combination treatment suppressed Akt/FOXO signaling and resulted in inhibition of key enzymes such as PKM2, LDH-A, ALDOA and HK1 for the Warburg effect. Overall, our study suggests that inhibition of RAC1 overcomes cisplatin resistance in ESCC both in vitro and in vivo, and provides a novel mechanism of chemo-resistance in ESCC. Therefore, targeting RAC1 might be a promising and potent strategy for treating ESCC in clinical practice.

Citation Format: Ruijie Zeng, Chunwen Zheng, Jinge Gu, Liu Peng, Haixia Zhang, Pan Feng, Yang Chen, Xiue Xu, Liyan Xu, Enmin Li. Overcoming cisplatin resistance of esophageal squamous cell carcinoma by targeting RAC1-regulated Warburg effect [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 4751.
Abstract 4754: CHTOP is a novel therapeutic target for chemoresistant epithelial ovarian cancer therapy
Authors: Bai Xupeng , Feng Xiaojie , Ni Jie , Beretov Julia , Deng Junli , Zhu Ying , Graham Peter , Li Yong
Published: August 2019

Background: Chemotherapy is the mainstay treatment for ovarian cancer (OC). Chemoresistance is a major challenge in epithelial ovarian cancer (EOC) therapy. CHTOP was identified as a potential chemoresistant biomarker in chemoresistant EOC cell lines using label-free LC-MS/MS proteomic technique. However, the role of CHTOP in EOC chemoresistance is still unclear.

Aim: In this study, we aimed to investigate whether CHTOP can be used as a therapeutic target in chemoresistant EOC cells and to reveal the mechanism underlying chemosensitization.

Methods: The expression difference of CHTOP was detected in chemoresistant and metastatic EOC cell lines by immunofluorescence (IF) and Western blot (WB). The expression of CHTOP in human EOC tissues was examined using immunohistochemistry (IHC). The effect of CHTOP knockdown (KD) on metastasis was examined using the Transwell® matrigel invasion and wound healing assays. Flow cytometry and TUNEL assay were employed to determine the association of CHTOP with apoptosis, while mammary sphere formation assay and IF were used to evaluate its regulation on EOC-cis cell stemness.

Results: The higher expression of CHTOP was found in EOC-cis (A2780-cis and IGROV-1-cis) and metastatic EOC (SKOV-3 and OV-90) cells as compared to normal epithelial ovarian cells (HOSE) by IF and WB. Also, high expression of CHTOP was found in human EOC tissues and associated with poor prognosis in patients. In contrast, CHTOP KD significantly reduced the metastatic potential of EOC-cis cells and increased their apoptosis at the presence of cisplatin. Furthermore, CHTOP KD decreased the stemness of EOC-cis cells.

Conclusion: Our findings suggest that CHTOP is associated with metastasis, apoptosis, and stemness in chemoresistant EOC cells, the survival in EOC patients, and might be a promising therapeutic target to overcome chemoresistance in EOC treatment.

Keywords: ovarian cancer, chemoresistance, CHTOP, metastasis, apoptosis, stemness

Citation Format: Xupeng Bai, Xiaojie Feng, Jie Ni, Julia Beretov, Junli Deng, Ying Zhu, Peter Graham, Yong Li. CHTOP is a novel therapeutic target for chemoresistant epithelial ovarian 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 4754.
Abstract 4756: Activation of AURKA contributes to TKI resistance in EGFR-mutant non-small lung cancer
Authors: Lee Meng-Hsuan , Hong Shiao-Ya , Kao Yu-Rung , Wu Cheng-Wen
Published: August 2019

The epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been widely used to treat EGFR-mutant non-small cell lung cancer (NSCLC) in clinics. Several generations of TKI were invented to overcome the unfavorable response and acquired resistance after long-term usage of EGFR TKI. Several mechanisms of acquired resistance have been reported. The well-studied resistant mechanism results from EGFR secondary mutations including T790M and C797S, which are involved in the resistance to first and third generation TKI, respectively. Other mechanisms aside from secondary mutation on EGFR including Her2 and MET amplification or epithelial mesenchymal transition (EMT) also distribute to TKI resistance. However, the resistant mechanisms which are independent to these alterations have not been completely identified yet. Here, we generated resistant cells selected by three different generations of TKI and analyzed their gene expression profiles. We discovered that many signal pathways, especially involved in cell cycle regulation, are commonly upregulated in three TKI-resistant cell lines. Aurora kinase A (AURKA) was the upstream kinase of those pathways; however, neither RNA level nor protein level of AURKA was significant changed. Interestingly, we found that phosphorylation of Aurora A at Thr288 in its catalytic domain is higher in TKI resistant cells. This reveals the possibilities that AURKA activation plays a role in the occurrence of TKI-resistance and that TKI-resistant NSCLC patients have a better response rate to AURKA inhibitor treatments.

Citation Format: Meng-Hsuan Lee, Shiao-Ya Hong, Yu-Rung Kao, Cheng-Wen Wu. Activation of AURKA contributes to TKI resistance in EGFR-mutant non-small 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 4756.
Abstract 4759: Antitumor activity of eribulin after fulvestrant plus palbociclib treatments in the HR(+) human breast PDx model
Authors: Asano Makoto , Nakagawa Takayuki , Semba Taro , Yokoi Akira , Matsui Junji , Funahashi Yasuhiro
Published: August 2019

Fulvestrant (FLV) and Palbociclib (Palb), which have an effect on degradation of estrogen receptor (ER) and inhibition of cyclin dependent kinases 4 and 6 (CDK4/6), respectively are used for treatments to postmenopausal breast cancer patients. However, there is not a clinical rationale to define a standard treatment regimen against breast cancer patients who are resistant to endocrine therapy plus Palb. Eribulin is a fully synthetic analog of the marine sponge natural product halichondrin B. Its clinical formulation is currently approved for advanced breast cancer (BC) and advanced liposarcoma in numerous countries. We examined the antitumor activity of eribulin after endocrine therapy (combination of FLV and Palb) compared with that of Capecitabine (Cape) in the luminal type breast cancer PDx model (OD-BRE-0438). OD-BRE-0438 is a patient derived xenograft (PDx) model developed from patient with luminal B breast cancer. Gene expression analysis by RNA-Seq showed that OD-BRE-0438 highly expressed ER and PR, but not ERBB2. OD-BRE-0438 tumor bearing mice were treated for 2 weeks of FLV (5mg/ head, Q7Dx2, sc) and Palb (10mg/kg, Q1Dx14, po), and treated mice were randomly divided using tumor volume around 350 mm3 as an index (day1). After that, mice were treated with eribulin (0.5 or 1 mg/kg, which are 1/4 MTD or 1/2 MTD in this model, Q7Dx2, iv) or Cape (125 or 250 mg/kg, which are 1/2 MTD or MTD in this model, Q1Dx14, po for 2 weeks). We measured tumor volume of each mouse after treatments (day 15) and analyzed the ratio of tumor volume at day 15 divided by that at day 1 (RTV: relative tumor volume). Treatments of FLV and Palb nearly completely inhibited in vivo growth of OD-BRE-0438 tumors, but didn’t cause tumor shrinkage. After two weeks of combination treatment with endocrine therapy, we started treatments with either eribulin or Cape, and compared the antitumor activity. Both drug showed strong antitumor activity with tumor shrinkage. In comparison of antitumor activity with either eribulin or Cape, that of eribulin was significantly greater than Cape. [eribulin 0.5mg/kg (RTV=0.42) vs Cape 125mg/kg (RTV=0.62, p=0.0068), eribulin 1.0mg/kg (RTV=0.32) vs Cape 250mg/kg (RTV=0.59, p=0.0008)]. Further investigation for mechanism of action of eribulin to show clear antitumor activity after treatments of FLV and Palb in the luminal type breast cancer PDx model are warranted.

Citation Format: Makoto Asano, Takayuki Nakagawa, Taro Semba, Akira Yokoi, Junji Matsui, Yasuhiro Funahashi. Antitumor activity of eribulin after fulvestrant plus palbociclib treatments in the HR(+) human breast PDx model [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 4759.