bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B019: Developing new ceramide analogs against AIDS-related lymphoma
Authors: Dai Lu , Foroozesh Maryam , Qin Zhiqiang
Published: August 2018

The Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of several human cancers, including primary effusion lymphoma (PEL), a subtype of highly aggressive non-Hodgkin lymphoma (NHL). PEL preferentially arises in immunocompromised patients, e.g., AIDS patients, but lacks of effective therapeutic options. Ceramide serves as a central mediator in sphingolipid metabolism and is referred to as a “tumor suppressor lipid”; however, its functional role in PEL survival and therapeutic value remain largely unknown. Our previous work demonstrated that targeting sphingolipid metabolism by either sphingosine kinase 2 (SphK2) inhibitor or exogenous ceramides induced significant PEL apoptosis and suppressed tumor progression. Recently, we have synthesized a series of new ceramide analogs with better stability and solubility. Our first screening indicated that at least 2 new compounds effectively inhibited PEL cell growth, through the complex mechanisms including induction of tumor cell apoptosis, cell cycle arrest, intracellular ceramide production, and viral lytic gene expression. By using an immunodeficiency mice xenograft model, we found that these selective ceramide analogs significantly repressed PEL progression in vivo. Ongoing work includes the identification of global gene profile affected by these new compounds within PEL cells using the NGS technology. We hope these new ceramide analogs can be developed as promising therapeutic strategies for prolonging the survival time of PEL patients and/or improving their life quality.

Citation Format: Lu Dai, Maryam Foroozesh, Zhiqiang Qin. Developing new ceramide analogs against AIDS-related lymphoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B019.
Abstract B029: Deletion of mesothelin impairs intraperitoneal growth of pancreatic cancer
Authors: Alewine Christine Campo , Rudloff Michael , Arons Danielle , El-Behaedi Salma , Zhang Xianyu , Avula Leela
Published: August 2018

The mesothelin (MSLN) protein is expressed in at least 85% of pancreatic adenocarcinomas but not on cells of the healthy pancreas nor in the parenchyma of other vital organs. Due to this differential expression, MSLN has been used as a target for various antibody-based treatments and cancer vaccines. The physiologic role of MSLN is unknown. The MSLN gene encodes for a precursor protein, which is cleaved to produce the mature MSLN protein. Previous reports have suggested that overexpression of MSLN precursor protein may increase tumorigenicity and metastatic potential of pancreatic adenocarcinoma. Here, we have used CRISPR-Cas9 gene editing to delete MSLN from the KLM-1 pancreatic cancer cell line (ΔMSLN) and have examined the growth of these cells compared to mock deleted cells (Mock). The ΔMSLN and Mock cells grew at the same rate both in cell culture and as subcutaneous tumors in nude mice, but had significantly impaired growth when inoculated into the mouse intraperitoneal cavity. Lentivirus transduction was used to restore expression of MSLN in ΔMSLN cells. Transduction of full-length WT MSLN, but not a Y318A mutant, restored intraperitoneal growth. Interestingly, reintroduction of mature MSLN instead of the precursor protein did not restore growth even though it was appropriately expressed on the cell surface. In summary, we have demonstrated that MSLN enhances the growth of pancreatic cancer within the peritoneal cavity and defined key regions of MSLN important for this activity.

Citation Format: Christine Campo Alewine, Michael Rudloff, Danielle Arons, Salma El-Behaedi, Xianyu Zhang, Leela Avula. Deletion of mesothelin impairs intraperitoneal growth of pancreatic cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B029.
Abstract B036: Synthetic DNA-encoded monoclonal antibody delivery (DMAbTM) of anti-CTLA4 antibodies induces tumor shrinkage in vivo
Authors: Duperret Elizabeth , Trautz Aspen , Patel Ami , Kim Joseph , Muthumani Kar , Weiner David B.
Published: August 2018

Antibody-based immune therapies targeting the T-cell checkpoint inhibitors CTLA4 and PD1 have made incredible advances in cancer therapy. In particular, the combination of CTLA4 and PD1 blockade (using ipilimumab and nivolumab, respectively) was recently shown to be the most effective immune therapy for improving progression-free survival in advanced melanoma patients. However, this immune therapy combination strategy is extraordinarily expensive due to high manufacturing costs of monoclonal antibodies, with an estimated annual cost of almost one million dollars per patient. In this study, we focused on the development of a DNA-encoded monoclonal antibody (DMAbTM) approach for delivery of anti-CTLA4 monoclonal antibodies in vivo. With this technology, formulated DMAbTM plasmids are injected into the muscle with electroporation, allowing for muscle cells to produce and secrete the DMAbTM for a prolonged period of time without the need for repeated administration. In proof-of-concept studies in mice, we show that delivery of a DMAbTM plasmid for a monoclonal antibody targeting mouse CTLA4 (clone 9D9) elicits high serum expression (peak expression of 7.9µg/mL in immune-competent C57Bl/6 mice with one injection of DNA). This anti-mouse CTLA4 DMAbTM is capable of inducing tumor regression in A/J mice that were implanted with the immunogenic Sa1N tumor cell line without repeated administration. Furthermore, this anti-mouse CTLA4 DMAbTM is capable of synergizing with a DNA vaccine targeting the tumor antigen TERT in slowing tumor growth for the nonimmunogenic TC-1 tumor cell line. We also examined DNA delivery of the anti-human CTLA4 antibodies ipilimumab and tremelimumab. For these anti-human CTLA4 DMAbsTM, we achieved steady-state levels of approximately 75µg/mL and 50ug/mL for ipilimumab and tremelimumab, respectively, in mice, which is greater than the mean trough levels of ipilimumab achieved in patients (21.8µg/mL serum concentration for 3mg/kg dose). These anti-human CTLA4 DMAbsTM produced in vivo bind to human CTLA4 protein and induce T-cell activation in a functional assay ex vivo. These results demonstrate the feasibility of delivering immune checkpoint blockade monoclonal antibodies using DNA for cancer immune therapy. Future studies will explore combination therapies with CTLA4 DMAbsTM and chemotherapy.

Citation Format: Elizabeth Duperret, Aspen Trautz, Ami Patel, Joseph Kim, Kar Muthumani, David B. Weiner. Synthetic DNA-encoded monoclonal antibody delivery (DMAbTM) of anti-CTLA4 antibodies induces tumor shrinkage in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B036.
Abstract B086: Targeting Auger electrons to cellular nuclei via PARP-1
Authors: Winters Harrison D. , Mach Robert H. , Mankoff David A. , Pryma Daniel A. , Makvandi Mehran
Published: August 2018

Introduction: Auger-emitting radionuclides have promising therapeutic properties due to their high linear energy transfer with nanometer ranges in tissue, although they have proven challenging to work with since they must be targeted to cellular nuclei to directly cause DNA damage. In the present study, we sought to utilize PARP-1 as a molecular target for the delivery of Auger electrons to induce local DNA damage without altering PARP-1 enzymatic activity. PARP-1 is an abundantly expressed nuclear protein involved in the DNA damage response and the primary target of emerging therapeutics for the treatment of homologous recombination deficient (HRD) ovarian cancers. Methods: In vitro cytotoxicity studies were performed by treating BRCA1-deficient ovarian carcinoma cell lines (UWB1.289 and OVCAR8) with various doses of [125I]KX1 or [123I]KX1, a PARP inhibitor analogue. To understand the specificity of KX1, the same treatments were given to matching cell lines that either were engineered to have a restored HR status via BRCA1 expression (UWB1.289 BRCA1-restored), or PARP1 knockout via CRISPR/cas9 genome editing (OVCAR8 PARP1-knockout). Next, cellular immunofluorescence was performed and DNA damage was assessed by measuring GH2AX, 53BP1, PAR, and PARP-1 levels at 2 or 24 hours after treatment with [125I]KX1 or [123I]KX1. Results: Cytotoxicity data revealed a dose-dependent reduction in cell viability in both BRCA1 HRD cell lines after treatment with either Auger emitter. Cell death in UWB1.289 BRCA1-restored and OVCAR8 PARP1-knockout cell lines was reduced when compared to identical treatments in parent cell lines. Immunofluorescence analysis showed a dose-dependent increase in GH2AX and 53BP1 foci as early as 2 hours post treatment in OVCAR8. Similar to the cytotoxicity data, GH2AX foci formation was reduced in OVCAR8 PARP1-knockout. Interestingly, treatment with radiolabeled [123I]KX1 did not reduce cellular ribosylation (PAR) as seen with the treatment of a commercially available PARP inhibitor, but resulted in an increase from control. Conclusions: Auger-emitting radionuclides delivered to nuclei via PARP-1 can cause significant DNA damage. Reduced cytotoxicity and GH2AX foci observed after treating OVCAR8 PARP1-knockout cell lines demonstrated that both Auger emitters caused DNA damage as a result of specifically targeting PARP-1. Furthermore, these agents did not inhibit the enzymatic activity of PARP-1 and may have the potential to elicit neoantigenicity through DNA damage without altering the proinflammatory pathways regulated by ribosylation.

Citation Format: Harrison D. Winters, Robert H. Mach, David A. Mankoff, Daniel A. Pryma, Mehran Makvandi. Targeting Auger electrons to cellular nuclei via PARP-1 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B086.
Abstract B207: Obesity and p16INK4A downregulation activate breast adipocytes and promote their pro-tumorigenicity
Authors: Alkhalaf Huda H.
Published: August 2018

Obesity is increasingly recognized as a risk factor for breast cancer development. However, the molecular basis of obesity-related breast carcinogenesis remains elusive. In this study, we have shown that obesity reduces the level of the tumor suppressor p16INK4A protein in breast adipocytes, which showed active features and strong pro-carcinogenic potential both in vitro and in orthotopic tumor xenografts, as compared to mature adipocytes from lean women. Furthermore, obesity triggered epithelial-to-mesenchymal transition (EMT) in breast ductal epithelial cells. Interestingly, specific downregulation of p16INK4A increased the expression/secretion levels of various adipokines including leptin, and activated breast adipocytes from lean women. Consequently, like breast adipocytes from obese women, p16-deficient adipocytes induced EMT in normal primary breast luminal cells in a leptin-dependent manner and enhanced tumor growth. Additionally, we have shown that p16INK4A negatively controls leptin at the mRNA level through miR-141 and miR-146b-5p, which bind the leptin mRNA at specific sequence in the 3’UTR. These results show that obesity activates breast stromal adipocytes through p16 downregulation, which upregulates leptin and promotes pro-carcinogenic processes.

Citation Format: Huda H. Alkhalaf. Obesity and p16INK4A downregulation activate breast adipocytes and promote their pro-tumorigenicity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B207.
Abstract KN03: Keynote Lecture: Cell transfer immunotherapy targeting unique somatic mutations in cancer
Authors: Rosenberg Steven A.
Published: August 2018

Adoptive cell transfer (ACT) immunotherapy using T lymphocytes with antitumor activity is a living therapy that can be highly effective in patients with a variety of metastatic cancers. Naturally occurring tumor-infiltrating lymphocytes (TIL) expanded in vitro and administered following a lymphodepleting preparative regimen mediated objective regression of metastatic melanoma in 56% of 194 patients, including complete regressions in 20% of patients who remain ongoing disease-free 3 to 10 years later. To determine the antigens recognized by TIL we developed an approach based on deep exomic sequencing of the cancer and immunologic testing of TIL or peripheral lymphocytes to generate T cells that recognized immunogenic mutations. TIL from 34 patients with metastatic melanoma recognized 78 random somatic mutations, none of which were shared among different melanomas. We next extended these studies to patients with common epithelial cancers and showed that 64 of 84 (70%) patients with a variety of human cancer types, including esophageal, colorectal, bile duct, gastric, pancreatic, ovarian, cervical, and lung cancer, contained T cells that recognized 142 neoantigens, all of which were unique except for KRAS (3 patients) and p53 (4 patients). Targeting unique cancer mutations has extended the reach of ACT immunotherapy and was used to mediate objective regressions in selected patients with chemorefractory metastatic cancers of the bile duct, colon, cervix, and breast. In addition to the use of naturally occurring mutation-reactive cells, we have genetically engineered autologous lymphocytes to express antitumor T-cell receptors (TCR) or chimeric antigen receptors (CAR) for use in ACT immunotherapy that can mediate durable cancer regressions in heavily pretreated patients with refractory lymphomas, sarcomas, and melanoma. Autologous T-cells can be used to provide a highly personalized immunotherapy for cancer patients refractory to conventional cancer treatments.

Citation Format: Steven A. Rosenberg. Keynote Lecture: Cell transfer immunotherapy targeting unique somatic mutations in cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr KN03.
Abstract LB-A18: Targeting the WNT-pathway in 3D-cultured in vitro PDX models for pancreas cancer
Authors: Dijkmans Thomas , Herpers Bram , Basten Sander , Yan Kuan , Giesemann Torsten , Schueler Julia , Price Leo
Published: August 2018

Creating patient-derived xenograft (PDX) models from human cancers allows bio-banking, propagation, characterization and drug testing on a large panel of tumor variants. To scale up the number of compound tests, identify relevant compound doses, increase the throughput, and study relevant cancer-driving pathways we developed 3D in vitro culture methods for PDX model-derived cell isolates. In this effort, each individual model is subjected to a wide panel of culture medium formulations, amongst which some media contain WNT-pathway stimulating components.

We identified an absolute dependency of a pancreatic in vitro PDX tumor to the WNT-signaling potentiating factor R-Spondin, while other pancreas tumors showed little or no dependency. To further study the dependency of pancreatic PDX tumors on the functionality and signaling of the WNT-pathway, we exposed in vitro PDX cultures to increasing doses of anti-FZD7, anti-FZD8 and to the Porcupine inhibitor LGK974, which blocks WNT palmitoylation and therefore secretion.

Our findings are that the WNT-pathway is functional and contributing to pancreas cancer outgrowth and morphological differentiation to different extents in different patient-models. Our in vitro PDX culture system in combination with 3D phenotypic analysis allows in-detail study of the WNT-pathway, as well as by supplementation or omission of secreted factors, addition of small molecule inhibitors and by exposure to antibodies. These in vitro PDX models can be used to test novel compounds and antibodies targeting the WNT-pathway and underscore the WNT-pathway as a potential target in pancreatic cancer.

Citation Format: Thomas Dijkmans, Bram Herpers, Sander Basten, Kuan Yan, Torsten Giesemann, Julia Schueler, Leo Price. Targeting the WNT-pathway in 3D-cultured in vitro PDX models for pancreas cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A18.
Abstract 1059: EpCAM differentially expressed on circulating and disseminated tumor cells promotes tumor metastasis in breast cancer patients
Authors: Lin Peter P. , Li Junjian , Wang Daisy , Gires Olivier , Wang Hongxia
Published: August 2018

To investigate the role of EpCAM expressed on both circulating tumor cells (CTCs) in peripheral blood and disseminated tumor cells (DTCs) in bone marrow with respect to tumor metastases, the integrated subtraction enrichment and immunostaining-FISH (SE-iFISH) was performed to quantify, phenotypically and karyotypically characterize cytogenetically abnormal CTCs and DTCs expressing EpCAM in breast cancer patients. In a cohort of 34 recruited late stage patients, consisting of 32 metastatic and 2 non-metastatic subjects, the average number of detected CTCs in metastatic vs non-metastatic patients was 25 and 18 cells, respectively. Among those CTCs in metastatic subjects, 23.4% of them showed EpCAM+, whereas none of EpCAM+ CTCs was detected in non-metastatic patients. There were total of 49 CTC clusters detected in metastatic patients, and 33% of those clusters were EpCAM+, however, only 3 CTC clusters were found in non-metastatic patients, and none of them was EpCAM+. In the case of DTCs, the average number of 2224 and 364 DTCs per subject was detected in metastatic vs non-metastatic subjects, with EpCAM+ ratio of 66.53% in metastatic and 8% in non-metastatic patients. Analysis of DTC clusters indicated that the average of 226 and 21 DTC clusters/subject were identified in metastatic and non-metastatic patients, respectively, and metastatic subjects showed 62% of DTC clusters EpCAM+, whereas 37% of clusters were EpCAM+ in non-metastatic patients. Comparing to the breast cancer serum biomarker CA15-3 detected in 32 metastatic cancer patients, showing positivity (≥28 ng/ml) of only 41% (13 out of 32), both CTCs (≥5) and DTCs (≥5) had a positivity rate of 81 (26 out of 32) and 97% (31 out of 32), respectively. Obtained results indicated that EpCAM+ CTCs and DTCs are reliable markers in terms of evaluating therapeutic efficacy and predicting cancer patients' prognosis.

Citation Format: Peter P. Lin, Junjian Li, Daisy Wang, Olivier Gires, Hongxia Wang. EpCAM differentially expressed on circulating and disseminated tumor cells promotes tumor metastasis in breast cancer patients [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 1059.
Abstract 1255: Delta-tocopherol induced endoplasmic reticulum stress causes autophagic degradation of ER and cell death in bladder cancer models
Authors: Blair Christopher A. , Hu Hanze , Huynh Tim , Wu Maggie , Yang Chung S. , Zi Xiaolin
Published: August 2018

Vitamin E has been the subject of numerous basic and clinical studies, which have demonstrated a range of results that necessitate the further analysis of its individual components. We demonstrate that of the major tocopherol components of Vitamin E, Delta tocopherol (δ-T) is significantly more effective than the more common Alpha- and Gamma- tocopherols at inducing bladder cancer cell death in vitro and reduces tumor growth and burden in vivo. As a major site of protein synthesis and folding the endoplasmic reticulum (ER) is highly sensitive to stress induced by increased unfolded protein load, which induces both pro-survival and ultimately pro-apoptotic responses via the different branches of the unfolded protein response (UPR). δ-T induces the UPR in human bladder cancer cells and ultimately results in cell death, inducing both apoptosis and autophagic cell death. Analysis of bladder cancer cells treated with δ-T revealed strong evidence of UPR activation in the form of upregulation and activation of ER stress sensors PERK and IRE1α, and the upregulation of folding chaperone GRP78 and transcription factors ATF4 and CHOP. These changes in turn induced downstream effects including Death Receptor 5 (DR5)-mediated apoptosis and visible induction of autophagy. In addition to the observation of autophagosome formation, induction of autophagy was further confirmed by upregulation of protein expression of components of the autophagic machinery LC3B and Atg family members. Electron Microscope imaging of treated cells revealed the presence of ER-whorls within the large autophagosomes which were themselves visible under conventional light microscopy, which has to our knowledge not been previously observed in cancer models. This observation is supported by the ER stress-based mechanism of action exhibited by δ-T on bladder cancer cells, which recapitulates the observations of the few previous reports of “ER-phagy” described in yeast models. ER stress induced expansion of the ER is believed to aid in the improvement of stressed cells' protein folding capacity, and selective degradation of the ER-whorls formed in this manner by a previously undescribed mechanism of autophagy was first described in yeast and remains unexplored in human cancer models. These findings may provide new insights into δ-T's mechanism of action and inform new approaches to the development of tocopherols as novel chemopreventive agents.

Citation Format: Christopher A. Blair, Hanze Hu, Tim Huynh, Maggie Wu, Chung S. Yang, Xiaolin Zi. Delta-tocopherol induced endoplasmic reticulum stress causes autophagic degradation of ER and cell death in bladder cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1255.
Abstract 1427: Comprehensive investigation of false mutation discoveries in FFPE samples
Authors: Blomquist Thomas M. , Xiao Wenming ,   Somatic Mutation Working Group of the SEQC2 Consortium
Published: August 2018

Next generation sequencing (NGS) has emerged as a primary tool for “precision” medicine, especially in the rapidly evolving field of cancer care. However NGS-based somatic variant calling in a clinical oncology setting is susceptible to numerous sources of technical error impeding optimal patient management. One important source of error in variant reporting is formalin fixation and paraffin embedding (FFPE). Of note, FFPE-derived errors are commonly present at clinically actionable loci and can occur at allelic frequencies (5-65%) that meet treatment criteria. Compounding this problem, degree of FFPE error is highly susceptible to inter-specimen and inter-laboratory variations. In this context, static variant calling thresholds to control for “general FFPE-effect” are likely inadequate; resulting in both False-Negatives (ignoring true somatic variation) and False-Positives (reporting technical error as truth). Despite the importance of fresh tissue for molecular studies, routine tissue histology (derived from FFPE) continues to be the gold standard for cancer staging/grading. And, as emphasis on smaller sample sizes becomes the norm, routine histology will be prioritized in many scenarios. Thus, the need to understand how molecular testing performs in FFPE tissue is a driving imperative for optimal cancer care.

In this study, our aim was to develop and evaluate “best” quality control practices that would minimize False-Positive or -Negative somatic variant calling in FFPE specimens. Reference samples (paired breast carcinoma and “normal” lymphoblast cell-lines) from the FDA-led Sequencing Quality Control Phase II (SEQC2) consortium were used. These specimens were subjected to varying FFPE conditions (n=8) mimicking the pathology laboratory setting. For each condition, multiple replicates of whole genome and exome sequencing (WGS/WES) were performed with ~100X coverage across the entire human genome. Observed somatic variation frequencies for each genomic position for each FFPE condition were compared to ground truth and quality control measures derived from matched fresh materials using numerous statistical models.

Applying rigorous analytical strategies, we aim to identify genomic regions that serve as markers (internal control regions) for the full range of FFPE-effect, develop QC measures and statistics to define lower limit of detection for variant calling for each genomic position, and provide general guidance for the community on how to accurately report mutations in FFPE processed clinical cancer specimens.

Citation Format: Thomas M. Blomquist, Wenming Xiao, Somatic Mutation Working Group of the SEQC2 Consortium. Comprehensive investigation of false mutation discoveries in FFPE samples [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 1427.
Abstract 1488: Inter-individual variation in hTERT regulation pathway genes in normal bronchial epithelial cells
Authors: Zolondek Rose , Craig Daniel J. , Crawford Erin L. , Willey James C.
Published: August 2018

Background: Genome-wide association studies (GWAS) and candidate gene studies suggest that telomere maintenance genes participate in etiology of lung cancer, idiopathic pulmonary fibrosis, and COPD. Telomeres are nucleoprotein structures located at the end of chromosomes that are maintained by telomerase encoded by hTERT. Telomerase is active in cancers, has the ability to aid in rapid cell division, and is dysfunctional in chronic conditions induced by inflammation as such as diabetes, renal failure, and COPD. hTERT is a known downstream target of β-catenin and WNT signaling. However, little is known regarding expression levels of these genes in normal bronchial epithelial cells (NBEC). The purpose of this study is to investigate inter-individual variation in regulation of β-catenin and WNT in NBEC, and if present, identify variants in regulatory region of human telomerase reverse transcriptase (hTERT) that could be used as candidate biomarkers for lung cancer risk. Methods: NBEC specimens from subjects with COPD (n=5) or without COPD (n=9) were obtained by bronchoscopy brush under IRB approved protocol. β-catenin, and WNT4 were measured by competitive multiplex-RT-PCR. Results: Both β-catenin and WNT4 displayed significant inter-individual variation as measured by Intra-Class Correlation (ICC) analysis (β-catenin ICC = 0.47, 95% Confidence Interval (0.2, 0.55), p < 0.05; WNT4 ICC = 0.60, 95% Confidence Interval (0.35, 0.85), p < 0.05). In patients with COPD, WNT4 expression was significantly higher in current smokers compared to former smokers (p<0.05). Conclusions: Inter-individual variation in NBEC expression of β-catenin and/or WNT4 was observed and it is reasonable to test the hypothesis that these genes and related pathway genes may contribute to variation in regulation of hTERT in NBEC and risk for lung cancer, COPD, and/or other lung diseases.

Citation Format: Rose Zolondek, Daniel J. Craig, Erin L. Crawford, James C. Willey. Inter-individual variation in hTERT regulation pathway genes in normal bronchial epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1488.
Abstract 167: Targeting the NF-kappaB pathway with bardoxolone methyl to inhibit ovarian cancer spheroid formation
Authors: Ozaki Michelle K. , House Carrie D. , Mazan-Mamczarz Krystyna , Lal-Nag Madhu , Thomas Craig , Annunziata Christina M.
Published: August 2018

Over 70% of patients with advanced stage ovarian cancer relapse within 2 years, with long term overall survival being only 25%. Tumor-initiating cells (TICs) are thought to contribute to this high rate of recurrence as they can resist killing by platinum-based chemotherapy. In this study we sought to identify pathways unique to TICs and to disrupt these pathways in order to more effectively target this population of cells. We conducted an siRNA screen which identified NF-kappaB signaling as an important pathway in the maintenance of ovarian cancer cells grown in non-adherent spheroid cultures that mimic TICs. Downregulation of several known NF-kappaB target genes, including NXF1, RAC2, CDC42, BCL2L1 and SERPINB3, significantly decreased growth of TIC cultures but not the corresponding adherent cultures, thereby indicating that expression of these genes is necessary for growth in TIC cultures. These findings are consistent with previous studies which showed that higher NF-kappaB activity is linked with drug resistance in patients. In parallel, we conducted a drug screen to identify compounds that target TICs. One clear candidate was bardoxolone methyl, which has been shown to inhibit IKK, an upstream NF-kappaB signaling protein. We confirmed the results of the drug screen in a panel of ovarian cancer cell lines, showing that bardoxolone methyl decreased known TIC markers CD133 and ALDH by flow cytometry, and decreased NF-kappaB proteins by western blot. To further elucidate the mechanism of bardoxolone methyl on TICs we conducted an RNAi screen +/- bardoxolone methyl. Interestingly, the knockdown of genes including BRAF, MAP3K8, LMNA and SMAD6 acted in a synthetically lethal manner with bardoxolone methyl in the TICs. To begin translating to a clinically relevant model, we proceeded to test whether bardoxolone methyl could eliminate TICs that remained after treatment with platinum-based chemotherapy. We confirmed in vitro that bardoxolone methyl in combination with chemotherapy reduced TIC populations. Our ongoing studies will address whether this drug is effective in preventing relapse in vivo after chemotherapy treatment of ovarian cancer in mouse models.

Citation Format: Michelle K. Ozaki, Carrie D. House, Krystyna Mazan-Mamczarz, Madhu Lal-Nag, Craig Thomas, Christina M. Annunziata. Targeting the NF-kappaB pathway with bardoxolone methyl to inhibit ovarian cancer spheroid formation [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 167.
Abstract 1749: CXCR4 inhibition modulates tumor microenvironment and robustly inhibits growth of B16-OVA melanoma
Authors: Saxena Ruchi , Wang Yan , Mier James W.
Published: August 2018

Background: The chemokine receptor CXCR4 is expressed on a range of immune cells. Additionally, CXCR4 has been found to be overexpressed in a variety of cancers and promotes cancer cell proliferation and metastasis, possibly by activating prosurvival signals that render cancer cells resistant to immune attack. Blockade of immune inhibitory pathways is emerging as an important therapeutic approach for the treatment of cancer. In our previous studies, X4-136, a CXCR4 antagonist, alone and in combination with various immune checkpoint inhibitor,s exhibited potent antitumor activity in the B16-OVA murine melanoma model. We report here results from additional in vivo studies as well as in vitro mechanistic experiments to determine the impact of CXCR4 inhibition on tumor cell gene expression and on immune phenotypes within the tumor microenvironment.

Methods: B16-OVA cells were implanted into C57BL/6 mice. Seven days post implantation, mice were treated with X4-136, anti-PD-L1, or the combination for 16 days. At sacrifice, tumors were excised and flash frozen in liquid nitrogen for Western blot analysis or treated with collagenase for the analysis of subpopulations of T-cells by FACS. Gene expression was determined by PCR and protein expression was analyzed by Western blot.

Results: X4-136 alone demonstrated more robust activity than anti-PD-L1. The antitumor activity of the anti-PD-L1 was further enhanced in combination with X4-136. Analysis of infiltrating immune cells by flow cytometry showed that X4-136 alone or in combination led to an increase of CD8+ T cells as well as CD8 and perforin double-positive T cells. A trend of decrease in both immunosuppressive populations, Treg and MDSC, was observed. This combination also led to a decrease in the expression of HIF-2α and cyclin D1 in comparison to tumors from vehicle-treated mice by Western blot analysis, suggesting an antiproliferative effect. Additional analysis of impacted genes and pathways will also be presented.

Conclusion: X4-136 alone increased tumor-infiltrating CD8+ T-cells and exhibited potent antitumor activity in the B16-OVA murine melanoma model. The enhanced antitumor activity was observed when X4-136 was added to anti-PD-L1 treatment. The activities observed were associated with the improved CD8+ T cells to Treg cells ratio and with improved tumor microenvironment.

Citation Format: Ruchi Saxena, Yan Wang, James W. Mier. CXCR4 inhibition modulates tumor microenvironment and robustly inhibits growth of B16-OVA 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 1749.
Abstract 1765: A humanized anti-CD47 monoclonal antibody that directly kills human tumor cells and has additional unique functional characteristics
Authors: Puro Robyn , Liu Katherine , Capoccia Benjamin , Donio Michael , Hiebsch Ronald , Bouchlaka Myriam , Carter Alun , Manning Pamela , Crowley Kathleen , Karr Robert
Published: August 2018

CD47 is a cell surface glycoprotein that interacts with signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells triggering a “don't eat me” signal that inhibits phagocytosis. Many tumors evade immune surveillance by overexpressing CD47, thereby preventing their recognition by phagocytes. Blocking the interaction of SIRPα/CD47 promotes phagocytosis and tumor cell destruction leading to a reduction in tumor burden.

We have developed a humanized anti-CD47 antibody, AO-176, that blocks the interaction between CD47 and SIRPα and exhibits several additional novel functional characteristics. These characteristics include the induction of cell death in multiple human tumor cell lines in a cell autonomous manner (not ADCC), assessed by an increase in phosphatidylserine/7AAD positive staining. A second novel characteristic is enhanced binding to tumor cells at acidic pH. AO-176 binds to human tumor cell lines in the high pM to low nM range at physiologic pH, however, binding is enhanced up to 20-fold at an acidic pH of 6.5. The acidic pH of the tumor microenvironment which ranges from 6.4-7.2 is characteristic of solid tumors and correlates with tumor progression and metastasis. As a result of this enhanced binding at acidic pH, AO-176 has the potential added advantage of tumor-specific targeting. A third novel characteristic exhibited by AO-176 is its selective binding to tumor cells while exhibiting reduced binding to normal cells including red blood cells (cynomolgus monkey and human), endothelial, epithelial and skeletal muscle cells. In addition to these novel characteristics, AO-176 also exhibits dose-dependent efficacy in multiple mouse tumor models.

Taken together, the unique combination of functional characteristics of AO-176, including induction of cell-autonomous killing, enhanced binding to tumor cells at acidic pH, significantly reduced binding to normal cells and potent in vivo efficacy provides the preclinical rationale for further development.

Citation Format: Robyn Puro, Katherine Liu, Benjamin Capoccia, Michael Donio, Ronald Hiebsch, Myriam Bouchlaka, Alun Carter, Pamela Manning, Kathleen Crowley, Robert Karr. A humanized anti-CD47 monoclonal antibody that directly kills human tumor cells and has additional unique functional characteristics [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 1765.
Abstract 1769: Antibody blockade of IL1RAP signaling reduces metastasis in a breast cancer model
Authors: Liberg David , Önnervik Per-Ola , Riva Matteo , Larsson Liselotte , Forsberg Göran , Wachenfeldt Karin von
Published: August 2018

Blockade of tumor inflammation has potential for cancer therapy, both as a primary mechanism to counter tumor growth but also in combination with other therapeutics. IL-1 signaling has been shown preclinically to be involved in tumor development and chemoresistance of pancreatic cancer, and blockade of IL-1 was recently shown to have a significant clinical impact on development of lung cancer. IL-1 receptor associated protein (IL1RAP) is a coreceptor for the IL-1 receptor (IL1R1) and is required for IL-1 signaling. IL1RAP is expressed in a number of tumor tissues, including lung and pancreatic cancer, both on tumor cells and on infiltrating immune cells. We have, using antibodies directed against IL1RAP, shown the ability to target and kill IL1RAP-expressing tumor cells by ADCC, to inhibit IL-1 signaling in those cells and to reduce growth of transplanted human tumors in vivo. To study effects of IL1RAP targeting on the tumor microenvironment and in an immune competent setting, an antibody towards mouse IL1RAP was generated. This antibody potently blocks mouse IL-1 (IC50 = 13 nM), binds to IL1RAP protein with high affinity (Kd = 4,2 nM), labels IL1RAP-expressing cells and can be administered to mice with good pharmacokinetics. In vivo imaging shows that the antibody is not generally distributed in tissues but localizes to tumor sites after injection. Treatment of mice with orthotopically implanted 4T1 breast cancer cells did not reduce primary tumor growth significantly but reduced both the number of (47% reduction, p=0.02) and size of lung metastases. Interestingly, 4T1 tumor cells express low levels of IL1RAP and are not responsive to IL1RAP blockade, but the effects instead relate to effects on the tumor microenvironment. We conclude that targeting of IL1RAP can, in addition to induce ADCC of tumor cells and block their response to IL-1, also inhibit metastasis by affecting the tumor microenvironment.

Citation Format: David Liberg, Per-Ola Önnervik, Matteo Riva, Liselotte Larsson, Göran Forsberg, Karin von Wachenfeldt. Antibody blockade of IL1RAP signaling reduces metastasis in a breast cancer model [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 1769.
Abstract 1796: Repurposing CRISPR to study aberrant AR signaling in models of castration-resistant prostate cancer
Authors: Kounatidou Evangelia E. , Robson Craig , Gaughan Luke
Published: August 2018

Prostate cancer (PC) is currently the most commonly diagnosed noncutaneous cancer affecting men worldwide. Despite the initial response to androgen ablation, patients relapse with tumors becoming refractory to clinically approved anti-androgens, resulting invariably in castration-resistant PC (CRPC). In CRPC, androgen receptor (AR) signaling is inappropriately restored. Resistance to available hormone therapies in PC has been linked to aberrant splicing of nascent AR transcripts and subsequent expression of constitutively active AR splice variants. Although we have information on the expression of receptor variants in advanced disease, the mechanisms that regulate these aberrantly functioning proteins are markedly understudied. Therefore, a better understanding of how splice variants are generated and function in this disease setting is critical for the development of more refined targeted therapies. By modeling clinically relevant AR splice variants, we will identify and exploit mechanisms of receptor regulation for advanced prostate cancer therapy. The CRISPR-Cas9 technology has been exploited to generate a CWR22Rv1 derivative cell line that lacks AR-FL and hence only expresses AR-Vs. This has allowed us to study the function of AR-Vs in an AR-FL-free background and therefore attribute the observed outcomes exclusively to AR splice variants. In addition, a novel CRISPR-based approach, currently developed in our lab, allows selective targeting of a catalytically inactive form of Cas9 (dead Cas9, dCas9) fused to a FLAG tag, to native chromatin regions and subsequent isolation of protein/RNA complexes associated with these sites. We have taken advantage of spatial mRNA splicing and transcription coupling in order to isolate splicing factors responsible for the generation of the AR variant 7 (AR-V7) in the CWR22Rv1 cell line and its AR-FL CRISPR knockout derivative. Three gRNAs were designed against the AR cryptic exon 3 genomic region. Targeting of FLAG-tagged dCas9 to that site was exploited for in situ capture and isolation of endogenous splicing factors specific to AR-V7, upon rapid FLAG immunoprecipitation and characterization by mass spectrometry. Identification of the AR-V7 spliceosome will fill a critical knowledge gap in our understanding of how AR-Vs are generated and provide key targets for future therapeutic intervention in advanced PC patients.

Citation Format: Evangelia E. Kounatidou, Craig Robson, Luke Gaughan. Repurposing CRISPR to study aberrant AR signaling in models of castration-resistant 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 1796.
Abstract 1849: Determining the molecular requirements for paclitaxel-induced spindle multipolarity
Authors: Scribano Christina
Published: August 2018

Paclitaxel (TaxolTM) and other microtubule poisons are standard chemotherapy agents used in the treatment of breast cancer. However, only a subset of patients experience positive therapeutic effects from these drugs, underscoring the importance of further mechanistic study. Recently we showed that paclitaxel does not accumulate in primary breast cancers at sufficient concentrations to cause mitotic arrest, as it does at typically used concentrations in cell culture. Instead, paclitaxel therapy induces multipolar spindles in breast cancers. Breast cancer cell lines treated with low nanomolar, clinically relevant doses of paclitaxel exhibit chromosome missegregation due to multipolar divisions. We have now extended this to demonstrate that low nanomolar concentrations of other clinically used microtubule poisons (including ixabepilone, vinorelbine, and eribulin) also induce multipolarity in breast cancer cells, suggesting that multipolar mitotic divisions may be a common mechanism of cytotoxicity for microtubule-targeted drugs. Importantly, supernumerary centrosomes are not required for microtubule poison-induced multipolar spindles. To gain mechanistic insight into the formation of paclitaxel-induced multipolar spindles, a variety of mitotic regulators were tested for their ability to affect spindle multipolarity in breast cancer cells treated with clinically relevant concentrations of paclitaxel. Our results demonstrate that the mitotic kinesin Eg5 and the kinase Plk1 promote both the formation and the maintenance of paclitaxel-induced spindle multipolarity, while the mitotic kinesin CENP-E and the kinase Mps1 do not. Interestingly, we found that decreasing the time to anaphase onset decreases the number of multipolar divisions in paclitaxel, resulting in reduced cytotoxicity. Previous studies have shown that Mps1 inhibition sensitizes cells to paclitaxel, and this combination therapy has entered clinical trials (NCT02138812 and NCT02366949). However, our evidence suggests that Mps1 inhibition reduces the efficacy of paclitaxel as a result of decreased time to anaphase onset and fewer multipolar divisions. Additionally, our results suggest that inhibitors of spindle pole clustering increase the number of paclitaxel-induced multipolar divisions and therefore have the potential to increase its efficacy. Identification of the cellular requirements for multipolarity induced by paclitaxel, and of agents that drive the metaphase-to-anaphase transition in the presence of multipolar spindles, may improve the clinical utility of paclitaxel and other microtubule poisons.

Citation Format: Christina Scribano. Determining the molecular requirements for paclitaxel-induced spindle multipolarity [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 1849.
Abstract 1857: Hydrophilic As4S4 nanoparticles inhibit K562 cell activity through ROS modulation and autophagy induction
Authors: Wang Tao , Wen Tao , Meng Jie , Liu Jian , Xu Haiyan
Published: August 2018

Realgar is crystalized As4S4 that has been demonstrated to be effective in treating hematologic malignancies by oral administration [1]. Nevertheless, the low bioavailability due to its poor solubility in neutral or acidic aqueous solution not only leads to the over dose administration of arsenic in clinical practice but also hinders the study of pharmaceutical mechanism for realgar. We previously reported a solid dispersion of realgar (e-As4S4), in which the crystal realgar (r-As4S4) was fabricated into nanoparticles encapsulated by the hydrophilic polymer using the hot melting co-extrusion technique [2]. The e-As4S4 exhibited much higher bioavailability and improved treatment efficacy in the acute myeloid leukemia (AML) animal model compared with r-As4S4.

This study investigated the effect of e-As4S4 has inhibitory on K562 cells and the underlying mechanisms. We showed that e-As4S4 down regulated the cellular ROS and HIF1-alpha in a concentration dependent manner. The low concentration of e-As4S4 induced both erythroid and megakaryocytic differentiation in the cells, evidenced by the up-regulation of CD235a and CD41 respectively, and the higher concentration induced cell cycle arrested in G2/M phase and apoptosis. Consistently, the proliferation rate and oxygen-consuming rate of the cells were reduced. Meanwhile the autophagy was detected when the cells were treated with e-As4S4, as well as the variation phosphorylation for p38 and Erk. These effects together induced the significant elimination of Bcr-Abl. In conclusion, e-As4S4 induced the bi-direction differentiation by modulating cellular ROS and inducing autophagy of the cells. These results therefore suggest that e-As4S4 represents a promising orally administrated drug for the treatment of CML.

Reference

1. Zhu HH et al. Oral tetra-arsenic tetra-sulfide formula versus intravenous arsenic trioxide as first-line treatment of acute promyelocytic leukemia: a multicenter randomized controlled trial. J Clin Oncol. 2013; 31:4215-21.

2. Ma Q et al. Fabrication of water-soluble polymer-encapsulated As4S4 to increase oral bioavailability and chemotherapeutic efficacy in AML mice. Sci Rep. 2016; 6: 29348.

Acknowledgement This work was supported by National Key R&D Program of China (2017YFA0205504) and CAMS Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-3-004).

Citation Format: Tao Wang, Tao Wen, Jie Meng, Jian Liu, Haiyan Xu. Hydrophilic As4S4 nanoparticles inhibit K562 cell activity through ROS modulation and autophagy induction [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 1857.