bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 782: Development of a high throughput in vitro screening platform to identify novel inducers of immunological cell death
Authors: Grillot Didier , Gangar Akanksha , Guillard-Huet Raphaelle , Boursier Eric , Potvain Florent , Serin Guillaume , Mirjolet Jean-François
Published: August 2018

Immunological cell death (ICD) is a form of cancer cell death induced by radiotherapy, photodynamic therapy and a few chemotherapeutic agents such as Doxorubicin, Mitoxantrone, and Oxaliplatin. Unlike apoptosis or necrosis, ICD can induce an effective immune response directed against the tumor whereby both dendritic cells and T lymphocytes are mediators of this response. Dying cancer cells recruit and activate immune cells by releasing damage-associated molecular patterns (DAMPS) that help and promote the immune response to antigenic tumor neo-epitopes. Three key DAMPS are associated with the ICD process: calreticulin exposition on the cell surface, ATP secretion and high-mobility group box 1 (HMGB1) release. In order to identify new therapeutic agents that promote ICD in malignant cells, we developed a screening strategy facilitated by an automated in vitro platform with four assays on three different tumor cell lines (human osteosarcoma U-2 OS, human breast MDA-MB-231 and murine liver Hepa 1-6). ICD inducers Doxorubicin and Mitoxantrone used as positive controls increased ATP secretion by 2 to 10-fold at a non-cytotoxic dose after 72 hours incubation on the three cell lines. Both compounds also increased calreticulin exposition by 2 to 4-fold (determined by immunofluorescence using the Operetta High-Content Imaging System) and HMGB1 release by two-fold on the three cell lines. Here we will present recent data from the screening of Oncodesign's Nanocyclix® library using this platform to identify novel ICD inducers.

Citation Format: Didier Grillot, Akanksha Gangar, Raphaelle Guillard-Huet, Eric Boursier, Florent Potvain, Guillaume Serin, Jean-François Mirjolet. Development of a high throughput in vitro screening platform to identify novel inducers of immunological cell death [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 782.
Abstract 785: Application of CRISPR/Cas9 gene editing to primary T cells
Authors: Li Xi , Tang Wanbing , Zhou Chenjie , Yang Yulin , Peng Zhengang , Zhou Wenrong , Ji Qunsheng , Cang Yong
Published: August 2018

T cells mediate a broad range of immune responses, especially generation of adaptive immunity for cancer cell clearance. Identification of genetic components that modulate T cell proliferation, differentiation, migration and cytotoxicity would facilitate development of more effective cancer immunotherapy. CRISPR/Cas9, as a tool, could generate gene knockout with high specificity and efficiency and has been widely used in genome-wide screening for therapeutic targets in many tumor cell models. However, its application to primary cells especially T cell has not been extensively investigated. Here, we described how to employ the CRISPR/Cas9 tools for immune-oncology target identification in primary mouse T cells. Cas9/sgRNA could be transduced into T cells with high efficiency and low toxicity using a lentivirus based delivery system. The transduced T cells acquired Cas9 expression but no DNA editing was observed. However, we achieved gene editing in primary T cells isolated from a Cas9 transgenic mouse strain after lentiviral delivery of guide RNA's. In summary, we provide a new method to generate DNA editing in primary T cell using CRISPR/Cas9 technology. It could be used for gene knockout, knockin and even genome-wide screening in primary T cell for immune checkpoint regulators.

Citation Format: Xi Li, Wanbing Tang, Chenjie Zhou, Yulin Yang, Zhengang Peng, Wenrong Zhou, Qunsheng Ji, Yong Cang. Application of CRISPR/Cas9 gene editing to primary T 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 785.
Abstract 80: Glucocorticoids mediate DCIS transition to IDC through myoepithelial cell apoptosis
Authors: Zubeldia-Plazaola Arantzazu , Recalde-Percaz Leire , Moragas Núria , Mancino Mario , Noguera Aleix , Puig Miquel Prats de , Guzman Flavia , Gascón Pedro , Bragado Paloma , Fuster Gemma
Published: August 2018

The most accepted theory in breast cancer evolution is the linear progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). DCIS is defined by a clonal proliferation of epithelial cells that accumulate within the lumen of a duct and surrounded by the myoepithelial cells layer and the basement membrane. The challenge is to predict which of the DCIS lesions will eventually progress to IDC and which will remain stable for a long time. Understanding the progression from DCIS to IDC is crucial to properly treat DCIS patients and to develop new and more approachable therapeutic strategies in this stage. The microenvironment, as well as stress factors like glucocorticoids, exerts strong influences in DCIS to IDC transition. These factors have been previously implicated in breast cancer progression however its role in the first stages of breast cancer and particularly in myoepithelial cells regulation remains elusive. In order to clarify glucocorticoids role in this breast cancer stage, we evaluated the effects of corticosterone and cortisol in epithelial cells and myoepithelial cells using in vitro 2D and 3D and in vivo techniques. Glucocorticoids induced a decreased in laminin levels and favour the disruption of the myoepithelial cell layer through myoepithelial cells apoptosis promotion. In addition, increased corticosterone levels, in an in vivo stress murine model, fostered the transition from DCIS to IDC via myoepithelial cell apoptosis and basement membrane vanishing. Furthermore, myoepithelial cell apoptosis is a more frequent event in DCIS+IDC than in DCIS breast cancer patients. Our findings show that stress, through glucocorticoids, plays a role in the progression of DCIS to IDC, and particularly by promoting apoptosis of myoepithelial cells and the apoptosis in myoepithelial cells seems to be a requirement to achieve invasive features in breast cancer patients.

Citation Format: Arantzazu Zubeldia-Plazaola, Leire Recalde-Percaz, Núria Moragas, Mario Mancino, Aleix Noguera, Miquel Prats de Puig, Flavia Guzman, Pedro Gascón, Paloma Bragado, Gemma Fuster. Glucocorticoids mediate DCIS transition to IDC through myoepithelial cell apoptosis [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 80.
Abstract 809: Afatinib restrains K-RAS driven lung tumorigenesis
Authors: Moll Herwig P. , Pranz Klemens , Musteanu Monica , Brcic Luka , Popper Helmut , Moldvay Judit , Dome Balázs , Casanova Emilio
Published: August 2018

Lung cancer is still the leading cause of cancer deaths worldwide. While it is well documented that patients suffering from epidermal growth factor receptor (EGFR) mutated non-small cell lung cancer (NSCLC) benefit from (EGFR) inhibition, there are still no successful targeted therapies for oncogenic mutations in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) gene available. Indeed, based on clinical trials using first generation EGFR tyrosine kinase inhibitors (TKI) it became a doctrine that K-RAS mutations drive resistance to EGFR inhibition in non-small cell lung cancer (NSCLC).

Conversely, we demonstrate here that EGFR signaling is engaged in K-RAS driven lung tumorigenesis both in human and in mice. Genetic mouse models revealed that deletion of Egfr quenches mutant K-ras activity and transiently reduces tumor growth. However, EGFR inhibition initiates a resistance mechanism which involves non-EGFR ERBB family members. This escape mechanism not only clarifies the disappointing outcome of first generation TKI in clinical trials, but also suggests therapeutic potential of pan-ERBB inhibitors. Indeed, by using several experimental models, including genetically engineered mouse models, (patient derived-) xenografts and in vitro experiments, we demonstrate that the FDA approved pan-ERBB inhibitor afatinib formidably reduces K-RAS driven lung tumorigenesis, whereas first generation TKI erlotinib and gefitinib remained ineffective in our models.

Altogether, our data strongly suggests revisiting the potential of EGFR TKI in clinical trials for the treatment of K-RAS mutated NSCLC, with a particular focus on pan-ERBB inhibitors, which are readily available.

Citation Format: Herwig P. Moll, Klemens Pranz, Monica Musteanu, Luka Brcic, Helmut Popper, Judit Moldvay, Balázs Dome, Emilio Casanova. Afatinib restrains K-RAS driven lung tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 809.
Abstract 821: B7-H3 is a potential antibody drug conjugate target for the treatment of prostate cancer
Authors: Davies Gareth C. , Ryan Kelli , Smith Alison J. , Lloyd Chris , Tammali Ravinder , Monks Noel , Lewis Arthur , Wilkinson Robert W.
Published: August 2018

B7-H3 is a member of the B7 family of immune-regulatory ligands and has been reported to be overexpressed in a broad range of human cancers. In order to understand more comprehensively the expression of B7-H3 on both normal tissues and on tumors, we developed an immunohistochemistry (IHC) assay for the detection of human B7-H3. Using this, we confirmed a restricted normal tissue expression pattern and demonstrated expression of B7-H3 in a range of solid tumors. In particular, a high proportion of prostate cancer samples showed strong homogenous B7-H3 expression. This low normal tissue expression and upregulation in cancer makes B7-H3 an attractive target for an antibody drug conjugate (ADC). To evaluate this approach, antibodies to B7-H3 were isolated by screening a phage display library for binding to B7-H3. These antibodies specifically bind to and are internalised by B7-H3 expressing cancer cells. ADCs were generated by site-specific conjugation of either a DNA-damaging pyrrolobenzodiazepine dimer or a microtubule-disrupting tubulysin payload. These ADCs selectively inhibited the growth of B7-H3 expressing cell lines including androgen-insensitive prostate cancer cell lines such as LNCAP-AI in vitro. Furthermore, these ADCs caused durable tumour regression in human tumor xenograft models in vivo. Together, these data suggest that an ADC targeting B7-H3 may have potential to be used for the treatment of a range of solid tumors including prostate cancer.

Citation Format: Gareth C. Davies, Kelli Ryan, Alison J. Smith, Chris Lloyd, Ravinder Tammali, Noel Monks, Arthur Lewis, Robert W. Wilkinson. B7-H3 is a potential antibody drug conjugate target for the treatment of prostate 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 821.
Abstract 83: The role of Nrf2-Keap1 pathway in the survival of circulating metastatic cancer cells
Authors: Aljohani Hashim M. , Furgason John M. , Amaya Peter , Deeb Ayham , Chalmers Jeffery J. , Bahassi El Mustapha
Published: August 2018

Approximately 90% of all cancer deaths arise from the metastatic spread of primary tumors. Of all the processes involved in carcinogenesis, local invasion and the formation of metastases are clinically the most relevant, but they are the least well understood at the molecular level. As a barrier to metastasis, cells normally undergo an apoptotic process known as “anoikis,” in circulation. The recent technological advances in the isolation and characterization of rare circulating tumor cells (CTCs) will allow a better understanding of anoikis resistance. Detailed molecular and functional analyses of anoikis-resistant cells may provide insight into the biology of cancer metastasis and help identify novel targets for prevention of cancer dissemination. To uncover the molecular changes that govern the transition from a primary lung tumor to a secondary metastasis and specifically the mechanisms by which CTCs survive in circulation, we carried out whole-genome sequencing (WGS) of normal lung, primary tumors and the corresponding brain metastases from five patients with progressive metastatic NSCLC. We also isolated CTCs from patients with metastatic cancer and subjected them to whole-genome amplification and Sanger sequencing and quantitative digital PCR of genes of interest. While the primary tumors showed mutations in genes associated with cell adhesion and motility, brain metastases acquired mutations in adaptive and cytoprotective genes involved in the response to cellular stress such as Keap1, Nrf2 and E300, which are key players of the Keap1-Nrf2-ARE survival pathway. Nrf2 is a transcriptional factor that upon stress translocates into the nucleus, binds to the ARE (anti-oxidant response elements) and drives the expression of antioxidant genes. The identified mutations affect regulatory domains in all three proteins, suggesting a functional role in providing a survival advantage to CTCs in the peripheral blood, allowing their dissemination to distant organs.

Citation Format: Hashim M. Aljohani, John M. Furgason, Peter Amaya, Ayham Deeb, Jeffery J. Chalmers, El Mustapha Bahassi. The role of Nrf2-Keap1 pathway in the survival of circulating metastatic cancer 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 83.
Abstract 837: Antitumor effects of anti-EGF antibodies generated by vaccination in NSCLC tumor cells
Authors: Garcia-Roman Silvia , Codony-Servat Jordi , Molina-Vila Miguel Angel , Bertran-Alamillo Jordi , Gimenez-Capitán Ana , Karachaliou Niki , d'Hondt Erik , Rosell Rafael
Published: August 2018

Background: Immunization against Epidermal Growth Factor (EGF) has demonstrated clinical efficacy in a phase III trial including unselected NSCLC patients. We are currently analyzing if anti-EGF antibodies generated by vaccination (anti-EGF VacAbs) showed antitumor activity in EGFR-mutant, Kras-mutant (mut) and Anaplastic Lymphoma Kinase (ALK) translocated non-small cell lung cancer cells (NSCLC), alone or in combination with tyrosine kinase inhibitors (TKI). In an EGFR-mut and in an ALK translocated cell lines we are also studying if the anti-EGF VacABs can delay the emergence of resistance to TKIs.

Methods: Anti-EGF VacAbs were obtained by immunizing rabbits with recombinant EGF. Cell lines were treated with anti-EGF VacABs alone and in combination with TKIs in EGFR (PC9, H1975) and ALK translocated (H3122, H2228) cell lines. In KRAS-mut cells (A549, H23) the combination was with chemotherapy agents. Cell viability was analyzed by MTT and apoptosis and cell cycle by fluorescence-activates cell sorting analysis (FACS). Changes of total and phosphorylated proteins were determined by Western blot. Sera from advanced NSCLC patients immunized with anti-EGF vaccine were also tested

Results: Anti-EGF VacAbs suppressed EGF-induced cell proliferation and inhibited downstream EGFR signaling in all cell lines tested. In combination, the anti-EGF VacAbs significantly enhanced the antitumor activity of gefitinib, erlotinib, osimertinib and afatinib in EGFR-mut cells, potentiated Erk ½ phosphorylation, arrested cell cycle progression and increased apoptosis. In addition, anti-EGF VacAbs blocked the activation of STAT3, downregulated the expression of proteins related to EGFR resistance, such as AXL, and significantly delayed the emergence in vitro of clones resistant to EGFR TKI. Sera from patients vaccinated against EGF also suppressed Erk ½ phosphorylation. Results for the combination of ALK TKIs and chemotherapy agents in ALK translocated and KRAS-mut cell lines will be presented at the meeting.

Conclusions: Anti-EGF VacAbs decreased cell proliferation and inhibited the activation of EGFR-pathway downstream proteins in EGFR-mut, KRAS-mut and ALK translocated cell lines. Also, they potentiate the effects of TKIs and prevent the emergence of resistance in EGFR-mut NSCLC cells. Two clinical trials are currently testing anti-EGF vaccination in EGFR-wt and EGFR-mut advanced NSCLC patients.

Citation Format: Silvia Garcia-Roman, Jordi Codony-Servat, Miguel Angel Molina-Vila, Jordi Bertran-Alamillo, Ana Gimenez-Capitán, Niki Karachaliou, Erik d'Hondt, Rafael Rosell. Antitumor effects of anti-EGF antibodies generated by vaccination in NSCLC tumor 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 837.
Abstract 839: Treatment and signaling contexts for the application of the imipridone ONC201 to prostate cancer cells
Authors: Amoroso Francesca , Pickard Adam , Glass Kimberly , Tarapore Rohinton , Allen J. E. , Mills Ian G.
Published: August 2018

Prostate cancer is the most common non-cutaneous cancer in men and a significant cause of cancer mortality. In the majority of the cases localised disease is treated with surgery or radiotherapy and whilst androgen deprivation therapy can extend survival in the subset of cases that progress, metastatic disease is incurable. We have previously reported that oncogenic drivers of prostate cancer (e.g. PTEN loss or mutation), as well as therapy, lead to changes in the activation of prostate cancer cell stress signalling pathways and in particular the unfolded protein response (UPR)1. Here we assess whether the activation of the UPR in response to radiation mirrors the immunogenic/inflammatory response, which is known to characterize the initially acute apoptotic phase before resolving to a chronic level. Further we have evaluated the impact of combining radiation with ONC201, a novel anti-cancer small molecule in clinical trials that is known to activate the pro-apoptotic stress response signalling2. We find that ONC201 has a delayed cytotoxic effect as a single agent in prostate cancer cells, with significant dose-dependent reductions in cell viability occurring 72 hours post-treatment. However activation of multiple arms of the UPR occurs earlier and is detectable at 24 hours for ATF4, ATF6 and IRE1-XBP1 at the protein/mRNA levels in the absence of impacts on cell viability. This delayed impact on viability offers a time window within which to evaluate short-term administration of ONC201 as a ‘primer' to enhance the cytotoxic response to radiation. We show that ‘priming' does indeed enhance radiotherapy response as assessed by cell viability and clonogenic assays. Since PTEN-loss is a high-incidence event in the development and progression of prostate cancer and has been linked to prognosis, we have also evaluated the impact of altering PTEN expression in our models as well as the effect of knocking down core regulators of the UPR. Cumulatively this work represents a further progression to positioning ONC201 in an appropriate molecular context to have durable effects when combined with other therapies to treat prostate cancer.

1.Sheng, X. et al., EMBO Molecular Medicine 7, 788-801 (2015).

2.Allen, J. et al. Discovery and clinical introduction of first-in-class imipridone ONC201. Oncotarget (2016). doi:10.18632/oncotarget.11814

Citation Format: Francesca Amoroso, Adam Pickard, Kimberly Glass, Rohinton Tarapore, J. E. Allen, Ian G. Mills. Treatment and signaling contexts for the application of the imipridone ONC201 to prostate cancer 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 839.
Abstract 84: Identification of HNRNPLL as a novel metastasis suppressor of colorectal cancer
Authors: Aoki Masahiro , Sasaki Eiichi , Kimura Kenya , Komori Koji , Shimizu Yasuhiro , Yatabe Yasushi , Sakuma Keiichiro
Published: August 2018

Despite the advances in surgery and chemotherapy for colorectal cancer patients, those with distant metastasis still suffer poor prognosis. Metastasis is a complex process regulated by a number of biomolecules, which can be conceptually divided into metastasis promoters and suppressors. To identify novel targets for prevention and/or therapy of colorectal cancer metastasis, we performed an in vivo shRNA library screen for colorectal cancer metastasis suppressor genes using an orthotopic transplantation model. CMT93 cells, a murine colorectal cancer cell line with poor metastasizing activity, were transduced with lentiviral shRNA library, and then transplanted into the rectum of C57/BL6 mice. Genomic DNA was collected from metastatic lesions, and the integrated shRNAs were retrieved by PCR for sequencing, followed by identification of the candidate genes targeted by the shRNAs. Among 47 candidate genes identified, we focused on Hnrnpll (heterogeneous nuclear ribonucleoprotein L-like) gene encoding a pre-mRNA binding protein involved in pre-mRNA splicing. Hnrnpll knockdown in CMT93 cells significantly increased their matrigel invasion in vitro and lung metastasis in vivo. An RNA-immunoprecipitation analysis identified Cd44 as a candidate target of Hnrnpll. The mRNA level of Cd44 variant 6 (Cd44v6), a splicing variant of Cd44 that had been shown to demarcate colon cancer stem cells driving colon cancer metastasis, was increased by knocking down Hnrnpll in colon cancer cells. Treatment with neutralizing anti-CD44v6 monoclonal antibodies suppressed matrigel invasion of colon cancer cells induced by Hnrnpll knockdown, suggesting that Hnrnpll may suppress invasion and metastasis, at least in part, by modulating the alternative splicing of Cd44. HNRNPLL expression level in colorectal cancer cells was drastically decreased upon induction of epithelial-mesenchymal transition (EMT), accompanied by increased expression of CD44v6. The link between HNRNPLL and EMT was further suggested by immunostaining analysis of clinical samples; colorectal cancer cells with low E-cadherin expression at the invasion front exhibited decreased HNRNPLL expression as compared with those in the tumor center. These results suggest that HNRNPLL is a novel metastasis suppressor in colorectal cancer. Possible mechanisms for regulation of HNRNPLL expression during EMT will also be discussed.

Citation Format: Masahiro Aoki, Eiichi Sasaki, Kenya Kimura, Koji Komori, Yasuhiro Shimizu, Yasushi Yatabe, Keiichiro Sakuma. Identification of HNRNPLL as a novel metastasis suppressor of colorectal 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 84.
Abstract 85: Oleic acid promotes head and neck squamous cell carcinoma anoikis resistance and metastasis via ANGPTL4/fibronectin pathway
Authors: Shen Chih-Jie , Chan Shih-Hung , Lee Chung-Ta , Huang Wan-Chen , Tsai Jhih-Peng , Chang Kwang-Yu , Chang Wen-Chang , Chen Ben-Kuen
Published: August 2018

Obese patients have higher levels of free fatty acids (FFAs) in their plasma and a higher risk of cancer than their non-obese counterparts. However, the mechanisms involved in the regulation of cancer metastasis by FFAs remain unclear. In this study, we found that oleic acid (OA) induced angiopoietin-like 4 (ANGPTL4) protein expression and secretion and conferred anoikis resistance to head and neck squamous cell carcinomas (HNSCCs). The autocrine production of OA-induced ANGPTL4 further promoted HNSCC migration and invasion. In addition, the expression of peroxisome proliferator-activated receptor (PPAR) was essential for the OA-induced ANGPTL4 expression and invasion. The levels of OA-induced epithelial-mesenchymal transition markers, such as vimentin, MMP-9, and fibronectin and its downstream effectors Rac1/Cdc42, were significantly reduced in ANGPTL4-depleted cells. Knocking down fibronectin not only inhibited the expression of MMP-9 but also repressed OA- and recombinant ANGPTL4-induced HNSCC invasion. On the other hand, ANGPTL4 siRNA inhibited OA-induced MMP-9 expression, which was reversed in fibronectin-overexpressing cells. Furthermore, the depletion of ANGPTL4 impeded the OA-primed metastatic seeding of tumor cells in the lungs. These results demonstrate that OA enhances HNSCC metastasis through the ANGPTL4/fibronectin/Rac1/Cdc42 and ANGPTL4/fibronectin/MMP-9 signaling axes. The inhibition of ANGPTL4 could be a potential strategy for the treatment of FFA-mediated HNSCC metastasis.

Citation Format: Chih-Jie Shen, Shih-Hung Chan, Chung-Ta Lee, Wan-Chen Huang, Jhih-Peng Tsai, Kwang-Yu Chang, Wen-Chang Chang, Ben-Kuen Chen. Oleic acid promotes head and neck squamous cell carcinoma anoikis resistance and metastasis via ANGPTL4/fibronectin pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 85.
Abstract 858: Enzalutamide versus abiraterone as a radiosensitizer in hormone-sensitive prostate cancer cells
Authors: Ghashghaei Maryam , Muanza Thierry , Alaoui-Jamali Moulay , Paliouras Miltiadis , Niazi Mohammad Tamim
Published: August 2018

Prostate cancer treatment is based on the estimated risk of recurrence in the U.S. Combined androgen deprivation therapy (ADT) with radiation therapy (XRT) is the standard of care for high-risk localized PCa. However, a large percentage of tumors are resistance to ADT due to continued AR signaling. Abiraterone (ABI), an androgen synthesis inhibitor, and Enzalutamide (ENZA), a potent AR antagonist, are new treatment options for metastatic castration resistance prostate cancer (mCRPC) patients. The aim of this study is to compare the efficacy of ENZA or ABI as a radiosensitizer in XRT therapy on PCa cells.

Methods: The effect of ENZA or ABI alone or in combination with XRT was assessed on hormone-sensitive (LNCaP, PC3-AR-T877A) and insensitive (PC3, PC3-AR V7) PCa cells using cell viability (MTT) and also clonogenic assays in different scheduling regimens: A- drug 24 h before XRT, B-drug 2h before XRT, or C- XRT followed by 24h later drug.

Results: We first determined the effect of ENZA or ABI on MTT assays in androgen-dependent (AD) and androgen-independent (AI) PCa cell lines. The results of MTT assay showed that ENZA inhibited the growth of the four different cell lines, LNCaP, PC3-T877A, PC3 and PC3 AR-V7 with IC50 values of 20, 22, 50, and 45μmol/L, respectively, after 24 hours of treatment. The same effect was observed on ABI treated cell lines. Radiosensitivity was not significantly increased in AD and AI PCa cell lines by ABI (DEF=1.00, in all cases) while there was a supra-additive dose enhancement factor (DEF= 1.75±0.08) for hormone-sensitive cells treated with ENZA (Table 1).

Conclusion: Our data indicates that ENZA acts as a much stronger radiosensitizer compared to ABI through different probable mechanisms of radiosensitivity.

Table 1. Dose Enhancement Factors calculations by scheduling ProtocolABCENZAABIENZAABIENZAABIADLNCaP1.35±0.021.05±0.011.75±0.081.001.30±0.051.00PC3-AR T877A1.30±0.031.001.65±0.011.001.35±0.061.05±0.02AIPC31.001.001.001.001.001.00PC3-AR V71.001.001.001.001.001.00

Citation Format: Maryam Ghashghaei, Thierry Muanza, Moulay Alaoui-Jamali, Miltiadis Paliouras, Mohammad Tamim Niazi. Enzalutamide versus abiraterone as a radiosensitizer in hormone-sensitive prostate cancer 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 858.
Abstract 863: Targeted α-therapy for treatment of metastatic melanoma
Authors: Nedrow Jessie , McCarty Greg , Cortez Angel , Roy Sanchita , Sgouros George
Published: August 2018

Background Very late antigen 4 (VLA-4; also called integrin α4β1) is overexpressed in melanoma tumors with an active role in tumor growth, angiogenesis, and metastasis. This makes VLA-4 an ideal antigenic target for targeted alpha therapy. The expression of VLA-4 on primary melanomas in human correlates with the development of metastasis and has been associated with high risk of metastasis and tumor progression. Furthermore, up-regulation of VLA-4 in melanoma is associated with a more aggressive, metastatic phenotype. Here in we evaluated an 225Ac-anti-VLA-4 conjugate as well as its 111In-labeled companion imaging agent for targeted radiotherapy in an aggressive mouse melanoma model. Methods An anti-VLA-4 antibody was conjugated to DOTA for 225Ac-labeling and DTPA for 111In-labeling. The resulting agents, 225Ac- or 111In-labeled anti-VLA-4 were evaluated in vitro, including binding affinity, internalization, and clonogenic assays. Furthermore, the therapeutic efficacy of 225Ac-anti-VLA-4 will be evaluated in multiple metastatic animal models of melanoma. Results 111In-DTPA-anti-VLA-4 demonstrated high affinity for VLA-4 in B16F10 cells, having a Kd of 0.18 ± 0.05 nM and approximately 46% was internalized at 6 hours. For delivery of an α-emitting radionuclide to VLA-4 positive tumor cells, the DOTA conjugate was labeled with 225Ac with specific activity of 3.5 MBq/nmol and >95% radiochemical purity, and demonstrated selective uptake in VLA-4 positive B16F10 cells. Clonogenic assays demonstrated a decrease in the surviving fraction of B16F10 cells treated with 225Ac-DOTA-anti-VLA-4 compared to controls. Therapeutic efficacy studies are currently underway. Conclusion 111In- and 225Ac-labeled anti-VLA-4 antibody conjugates are capable of selectively delivering radionuclides for SPECT imaging and targeted alpha therapy to melanoma tumor cells that overexpresses VLA-4. The agents presented here have the potential to identify and treat patients who have metastatic melanoma.

Citation Format: Jessie Nedrow, Greg McCarty, Angel Cortez, Sanchita Roy, George Sgouros. Targeted α-therapy for treatment of metastatic melanoma [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 863.
Abstract 870: Effects of epigenetic modifier inhibitors on AML cell sensitivity to differentiation therapy
Authors: Heimdal Kalsi K. , Hernandez Edjay Ralph A. , Super Heidi J. Gill
Published: August 2018

Human acute myeloid leukemia (AML), the most common type of acute leukemia, has approximately eight subtypes, many of which have poor prognosis. Most of these subtypes are associated with specific, recurrent chromosome translocations. These translocations result in fusion genes, which encode oncoproteins that block differentiation and promote proliferation of immature cells. The Myeloid Lymphoid Leukemia gene (MLL) is frequently involved in these translocations, and is considered a driver of the AML. Differentiation promoting drugs, such as all-trans-retinoic acid (ATRA) are an attractive alternative to cytotoxic chemotherapy, but few types of AML, other than acute promyelocytic leukemia (APL), respond to ATRA. We hypothesize that specific genes must be activated or inhibited in AML for drugs like ATRA to induce differentiation, and that gene activation or inhibition may be the result of specific epigenetic modification. We also hypothesize that AML with different genetic alterations may respond differently to specific epigenetic inhibitors. Our initial studies have focused on one AML cell line, MV4;11, with alterations in the MLL gene, and one non-MLL related AML cell line, U937. MV4;11 and U937 were treated with specific epigenetic modifiers, including tranylcypromine (TCP), an inhibitor of histone demethylase KMD1A/LSD1, and CI-994, a general histone deacetylase inhibitor. Evidence for differentiation was noted in a variety of assays, including reduced cell proliferation as measured directly and by MTT assay, upregulation of myeloid-specific cell surface markers such as CD11b as measured by fluorescence-activated cell sorting (FACS), and myeloid-related nuclear morphological changes noted with cytospin analysis of cells. Both CI-994 and TCP seemed to sensitize U937 cells to differentiate when treated with ATRA as measured by all indicators of differentiation. MV4;11 cells were sensitized to differentiation by CI-994 with or without ATRA, although there was no indication of CD11b upregulation. However, MV4;11 was not sensitized by TCP to differentiate with ATRA. These experiments suggest epigenetic inhibitors may increase sensitivity to differentiation therapy, but that the response may still be dependent on the specific genetic alteration driving the AML. Our studies suggest that in MLL-driven leukemia, the MLL fusion oncoprotein may override genetic manipulation resulting from treatment with epigenetic inhibitors, preventing differentiation by ATRA.

Citation Format: Kalsi K. Heimdal, Edjay Ralph A. Hernandez, Heidi J. Gill Super. Effects of epigenetic modifier inhibitors on AML cell sensitivity to differentiation therapy [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 870.
Abstract 887: The effect of anticancer chemotherapeutics on immune microenvironment of lung adenocarcinoma with EGFR mutation in vivomicroenvironment of lung adenocarcinoma with EGFR mutation in vivo
Authors: Suh Young-Ah , Mun Hyemin , Lee Sun-Hye , Jo Se-Young , Oh Ju-Hee , Lee Chu-Hee
Published: August 2018

Lung cancer is the leading cause of cancer related deaths worldwide. Molecular targeting therapy is an attractive treatment for lung cancer, although it eventually develops resistance in most cases. In this report, we tested taxan antitumor effect on lung adenocarcinomas in vitro and in vivo. It could inhibit cell growth of many NSCLC cells tested, exhibiting reduced expression of c-MET and STAT3. The nascent effect of taxan on lung tumor cells were also proved on patient-derived primary lung tumor cells and patient-derived xenograft model that highly expresses c-MET. The antitumor effect of this chemotherapeutics was also observed on T790M mutant cells. Interestingly, the antitumor effect of taxan was synergistic in combination of c-Met inhibitor on lung tumor cells as well as patient-derived primary lung tumor cells. However, single treatment of taxan was sufficient for halting tumor growth in vivo, as no combinational effect of c-Met inhibitor was obvious. Not only c-Met inhibitor but also taxan reduced phosphorylated c-MET and STAT3, although cytokines on downstream of c-MET and STAT3 were explosively increased in vitro setting resulting in high expression of chemokines/cytokines. Importantly, the effect was not true in in vivo setting, in which the expressions of chemokines/cytokines as well as phosphorylated c-MET and STAT3 were influenced with this agent. These results suggest that taxane is one of the comparative adjuvant for lung tumors exhibiting targeted drug resistance, especially for enhanced signaling of MET-STAT-immuno-microenvironment.

Citation Format: Young-Ah Suh, Hyemin Mun, Sun-Hye Lee, Se-Young Jo, Ju-Hee Oh, Chu-Hee Lee. The effect of anticancer chemotherapeutics on immune microenvironment of lung adenocarcinoma with EGFR mutation in vivomicroenvironment of lung adenocarcinoma with EGFR mutation in vivo [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 887.
Abstract 888: A novel assay for the evaluation of ADCC in 3D-culture
Authors: Irigoyen Macarena , Alms Andrea , Ejiri Yoko , Ayano Satoru , Castillo Gonzalo
Published: August 2018

In the last few years, one of the most innovative areas of cancer research has been immuno-oncology where significant advancements have been seen, particularly, in the use of monoclonal antibodies to treat cancers. The regulation of immune-mediated killing by monoclonal antibodies is an important mechanism of action, like in the case of trastuzumab, a humanized monoclonal antibody used in the treatment of HER-2 positive breast cancers. Binding of the humanized antibody to the receptor induces antibody-dependent cellular cytotoxicity (ADCC). Assays that monitor this activity are historically difficult to execute suffering from low sensitivity and reproducibility. Furthermore, experiments are typically done in suspension of cells grown as monolayers; often, showing limited clinical translatability when studying solid tumor models. Unencumbered by the limitations of 2D assays, the tumor microenvironment of 3D-cultures includes cell-cell interactions and formations of metabolic gradients, which are vital to understanding drug efficacy and resistance. To study the ADCC triggered by trastuzumab in 3D-cellular models, we developed a novel method to evaluate cellular toxicity in spheroids. In this study, we demonstrated the quantification, with specificity and precision, of the ADCC activity elicited by trastuzumab in 3D-culture. The high HER2-expressing BT474 cell line (target cells) was pre-labeled and formed into spheroids utilizing Kuraray's 384-well Elplasia's microplates. PBMCs (effector cells) isolated from healthy donors were added in the presence or absence of the antibody, trastuzumab, and incubated overnight. Cytotoxicity of the tumor-derived cells was evaluated by counting stained dead cells using a cell imager. In parallel, a 2D-platform for ADCC assay was run and the pharmacology of trastuzumab was characterized. BT474 cells grown as spheroids show an IC50 of 0.09 µg/mL, when the study was done in 2D-culture an IC50 of 0.028 µg/mL was obtained. The ability to evaluate ADCC in both 2D- and 3D- may help the development of more translatable models in the clinic.

Citation Format: Macarena Irigoyen, Andrea Alms, Yoko Ejiri, Satoru Ayano, Gonzalo Castillo. A novel assay for the evaluation of ADCC in 3D-culture [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 888.
Abstract 890: Calmodulin governs nuclear entry of fusion PAX3/FOXO1 oncoprotein, a target in alveolar rhabdomyosarcoma
Authors: Darvishi Emad , Thomas Cheryl Lynn , Gryder Berkley Eric , Shern John Frederick , Khan Javed , Woldemichael Girma M
Published: August 2018

Reciprocal chromosomal translocations resulting in the fusion of the DNA binding domain of the transcription factor PAX3 and the transactivational domain of FOXO1 underlie most cases of alveolar rhabdomyosarcoma (aRMS). Testing of compound MTPF63 against a panel of sarcoma cell lines showed that it had an inhibitory effect on proliferation of PAX3-FOXO1 translocation-positive cells. Further evaluation of its activity in Rh4 cells transduced with a PAX3-FOXO1 luciferase reporter showed that it attenuated reporter activity. Additional confirmation for the compound's inhibitory impact on PAX3-FOXO1 activity was obtained by detecting significant attenuation in the levels of Alk1 and MyoD1 proteins whose expression is driven by PAX3-FOXO1 in aRMS cells. Cell viability assays performed under 2D and 3D culturing conditions demonstrated that MTPF63 inhibits cell growth more effectively in translocation-positive alveolar RMS than in translocation-negative embryonal RMS. A hypothesis for MTPF63's mode of action was generated using the complete pool of barcoded essential heterozygous (~1150 strains) and homozygous (~4800 strains) diploid gene deletion mutants of Saccharomyces cerevisiae to identify gene deletions that confer sensitivity to the compound. Gene ontology enrichment analysis of 255 sensitive mutants whose growth rates were significantly inhibited in the presence of the compound compared to control, not only revealed calmodulin as main target of the compound but also illustrated the compound's interference with calmodulin-dependent nuclear protein import pathway, suggesting that it might impact PAX3-FOXO1 nuclear localization. Immunostaining of nuclear-localized PAX3-FOXO1 oncoprotein in two aRMS cell lines revealed cytoplasmic localization. Immunoblot analysis of fractionated cytosolic and nuclear protein lysates for PAX3-FOXO1 in MTPF63 treated cells also showed increased amounts in the cytosolic fractions. Transient overexpression of calmodulin was shown to partially rescue the inhibitory effect of the compound on PAX3-FOXO1-driven luciferase expression in RH4 reporter cells. Taken together, the data demonstrate the involvement of calmodulin in the nuclear import of PAX3-FOXO1 and introduce calmodulin antagonists as potential therapeutics against translocation-positive childhood alveolar rhabdomyosarcoma.

Citation Format: Emad Darvishi, Cheryl Lynn Thomas, Berkley Eric Gryder, John Frederick Shern, Javed Khan, Girma M Woldemichael. Calmodulin governs nuclear entry of fusion PAX3/FOXO1 oncoprotein, a target in alveolar rhabdomyosarcoma [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 890.
Abstract 896: Adjutant therapeutic strategy for sorafenib-resistant hepatocellular carcinoma
Authors: Peng Syue-Wei , Lin Yi-Heng , Teng Ming-Hao , Lai Hung-Cheng , Chang Te-Sheng , Huang Yen-Hua
Published: August 2018

Purpose: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is the leading cause of cancer mortality worldwide. To date, Sorafenib remains the standard of care for advanced HCCs not amenable to curative therapies such as surgery or local ablation. Unfortunately, clinical efficacy of Sorafenib remains marginal as most HCC patients receiving Sorafenib treatment develop Sorafenib resistance. The recently approved regorafenib is the only rescue therapy for patients who fail Sorafenib treatment. However, the effect of regorafenib is also far from satisfactory. Therefore, new strategies to overcome sorefenib resistance are critically warranted. Recently, one FDA-approved drug, here we tentatively named as “FA”, was shown to suppress different cancer malignancy by various mechanisms including inhibiting the development of drug resistance in cancer. These findings provide a rationale for combination treatment of this FA drug with Sorafenib in Sorafenib-resistant HCC. Results: Two Sorafenib-resistant HCC cell lines of Hep3B and HepG2215 were established. The Sorafenib resistance characteristics of the HCC cells were further verified by western blotting assay detecting protein tyrosine phosphorylation. We also demonstarted the resistant HCC cells expressed stemness-related genes and showed high ability of cell migration and metastasis. Combination use of FA drug and Sorafenib significantly suppressed the cell viability as well as the stemness-related properties of Sorafenib-resistant HCC cells, including stemness-related gene/protein expression, secondary sphere formation, and the cell migration. Conclusion: Combination use of FA drug and Sorafenib would be a potential therapeutic strategy for Sorafenib-resistant HCC treatment.

Citation Format: Syue-Wei Peng, Yi-Heng Lin, Ming-Hao Teng, Hung-Cheng Lai, Te-Sheng Chang, Yen-Hua Huang. Adjutant therapeutic strategy for sorafenib-resistant hepatocellular carcinoma [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 896.
Abstract 899: Phosphorylated mTOR by signaling through EphB3 confers resistance to a selective FGFR inhibitor in SNU-16 gastric cancer cells
Authors: Lee Suk-young , Na YooJin , Jung YoonA , Kim JungLim , Oh Sang Cheul , Lee Dae-Hee
Published: August 2018

Because about 10% of gastric cancers show amplification of FGFR2, inhibition of FGFR2 activation has been regarded as one of therapeutic targets. AZD4547, a selective inhibitor of the FGFR1-3 tyrosine kinases, was developed to inhibit FGFR signaling; however, its efficacy is limited by emergency of the acquired resistance. We tried to clarify a resistance mechanism against the FGFR inhibitor. The cell line resistant against AZD4547 was established using SNU-16 (SNU16R), an FGFR2-amplified gastric cancer cell line, by culturing with increasing concentration of AZD4547. The expression levels of FGFR or phosphorylated FGFR (pFGFR), EphB3 and downstream signaling molecules, proteins involved in epithelial-mesenchymal transition (EMT) and apoptosis, were determined by Western blot. Cell viability was measured with EZ-Cytox kit reagent (DoGen, Seoul, Korea). Relative level of tyrosine phosphorylation of receptor tyrosine kinases (RTKs) was evaluated with Proteome ProfilerTM array by human phosphor-RTK kit (R&D Systems). Apoptosis was determined by Annexin V-FITC apoptosis detection kit (BioBud, Seoul, Korea). Cell cycle assay was performed using Beckman Coulter flow cytometer and WinMDI for determination of cell numbers in G1, S, G2/M phases. Here we show the following: 1) establishment of a gastric cancer cell line (SNU16) resistant to AZD4547 (SNU16R); 2) expression of EphB3 receptor in SNU16R and induction of apoptosis and cell cycle arrest as well as inhibition of cell proliferation and suppressed expression of EMT markers under the treatment EphB3 inhibitor in SNU16R; 3) demonstration of mTOR as a downstream signaling cascade molecule of EphB3 by showing increased expression of phosphorylated (p-) mTOR and its downstream molecules in SNU16R and suppressed expression of p-mTOR by the EphB3 inhibitor. Cell cycle arrest, inhibition of cell proliferation and suppressed expression of p-mTOR under treatment with everolimus were also shown. We concluded that phosphorylation of mTOR through activation of EphB3 receptor is one of the mechanisms that confer resistance to AZD 4547, a selective FGFR inhibitor, in gastric cancer cells.

Citation Format: Suk-young Lee, YooJin Na, YoonA Jung, JungLim Kim, Sang Cheul Oh, Dae-Hee Lee. Phosphorylated mTOR by signaling through EphB3 confers resistance to a selective FGFR inhibitor in SNU-16 gastric cancer 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 899.