bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3757: Probing glioblastoma and its microenvironment at single cell resolution
Authors: Peng Sen , Rath Sanhita , Bollam Saumya , Eschbacher Jenny , Mehta Shwetal , Sanai Nader , Berens Michael , Kim Seungchan , Dhruv Harshil
Published: August 2019

Single-cell sequencing (scSeq) is a powerful tool to investigate cancer genomics at single cell resolution. Multiple studies have recently illuminated intratumoral heterogeneity in glioblastoma, however, the majority focused on molecular complexity of tumor cells, without taking into account unexplored host cell types that contribute to the microenvironment around GBM tumor. To address the glioblastoma microenvironment composition and potential tumor-host interactions, we performed deep coverage (176k average reads per cell) scSeq of freshly resected primary GBM patient tissue without implementing any tumor cell enrichment strategies. scSeq libraries for 902 cells were prepared using 10X Gemcode platform and sequenced on Illumina NextSeq 500. This run was of high quality with 2,663 median genes per cell and low mitochondrial gene percentage (median < 5%). We used Cell Ranger analysis pipelines and Seurat packages to classify individual cells into 10 clusters and visualize them using t-SNE two-dimensional projections. We then identified the signature gene set for each cluster, relative to all other cells. Pathway analysis of each cluster signature along with known GBM microenvironment cell signatures revealed glioma tumor population along with surrounding microglia/marcophages, astrocytes, pericytes, oligodendrocytes, T cells and endothelial cells. Cell type markers identified by single cell transcriptomics were validated by IHC analysis. Microenvironmental composition and single cell signature will be confirmed through single nuclei sequencing of preserved (Frozen) tumor sample. Our results demonstrate the cellular diversity of brain tumor microenvironment and lay a foundation to further investigate the individual tumor and host cell transcriptomes that are influenced not only by their cell identity but also by their interaction with surrounding microenvironment.

Citation Format: Sen Peng, Sanhita Rath, Saumya Bollam, Jenny Eschbacher, Shwetal Mehta, Nader Sanai, Michael Berens, Seungchan Kim, Harshil Dhruv. Probing glioblastoma and its microenvironment at single cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3757.
Abstract 3759: Host and neoplastic cell secretions of proteolytic enzymes potentiate metastasis
Authors: Dykes Samantha S. , Fasanya Henrietta O. , Siemann Dietmar W.
Published: August 2019

The presence of macrophages within breast tumors is associated with metastatic potential. Tumor-associated macrophages are often stimulated by Interleukin-4, resulting in a pro-tumorigenic (M2-like) phenotype and the secretion of growth factors and proteases, including the lysosomal protease cathepsin L (CTSL). CTSL is often secreted by breast cancer cells and stromal cells and contributes to tumor invasion, metastasis, and angiogenesis. We hypothesized that secretion of the proteolytic enzyme cathepsin L by both tumor-associated macrophages and neoplastic cells facilitate tumor invasion and dissemination. Initial studies found that inhibiting CTSL using the novel cathepsin L/K inhibitors KGP94 and KGP207 prevented in vitro M2 macrophage invasion and reduced macrophage-stimulated invasion of 4T1 murine breast cancer cells. Interestingly, KGP94 and KGP207 also reduced the expression of several M2-associated markers, suggesting that CTSL activity may be important for Interleukin-4-driven M0 to M2 differentiation. Furthermore, CTSL shRNA knockdowns revealed that CTSL supplied from both the tumor cell and the macrophage population is important for tumor cell invasion. These data suggest that tumor cells and macrophages may both contribute to the CTSL-driven metastatic phenotype of breast cancer. Taken together, these studies highlight the importance of CTSL in macrophage functions and suggest that cathepsin inhibition strategies may be therapeutically beneficial by impairing the progression of tumors with high infiltration of M2 macrophages.

Citation Format: Samantha S. Dykes, Henrietta O. Fasanya, Dietmar W. Siemann. Host and neoplastic cell secretions of proteolytic enzymes potentiate metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3759.
Abstract 3768: Single-cell characterization and lineage tracking of recurrent pediatric rhabdomyosarcoma
Authors: Patel Anand G. , Stewart Elizabeth , Clay Michael , Chen Xiang , Dyer Michael
Published: August 2019

A critical challenge for the effective treatment of pediatric solid tumors is tumor recurrence. In pediatric rhabdomyosarcoma (RMS), 70% of patients with metastatic disease will experience relapse despite treatment with chemotherapy, radiation and/or surgery. Those patients with relapse have particularly dismal outcomes with less than 20% survival over 5 years. These poor outcomes underscore the need for a better understanding of RMS, particularly the biologic processes responsible for recurrence and progression. Currently, a major obstacle towards improving RMS treatment has been that we, as a field, do not know which cells within RMS tumors evade treatment toxicity, and how these cells are selected for in the presence of chemotherapy.

We have previously developed a panel of orthotopic patient-derived xenografts (O-PDXs) that encompass the clinical and molecular diversity of RMS. In this study, we have leveraged single-cell RNA-sequencing (scRNA-seq) of our O-PDX system to characterize the intratumoral heterogeneity within this panel. Analysis of single-cell transcriptomic data demonstrate that fusion-negative RMS O-PDXs bear two distinct subpopulations: (1) a major subpopulation encompassing approximately 99% of the tumor cells, and (2) a minor population that are non-cycling and express genes from a variety of mesenchymal lineages. This two-population intratumoral structure is stable within O-PDXs during passaging of xenografts.

To extend these findings, we have developed a workflow using lentiviral transduction of O-PDX cells with a high complexity barcode library. This library integrates a unique 18-mer oligonucleotide barcode into each transduced cell within the 3’-untranslated region of blue fluorescent protein. Thus, all cells within an O-PDX with the same barcode are progeny of the same ancestral cell. We have validated longitudinal tracking of barcode diversity using genomic DNA and within the libraries generated during scRNA-seq. Mice bearing barcoded O-PDXs have undergone chemotherapy dosed to pharmacokinetically mimic pediatric therapy regimens; O-PDXs regress during treatment and ultimately recur following completion of therapy, akin to the clinical experience with RMS. Recurrent O-PDXs bear divergent barcodes, indicating that chemotherapy selects for tumor cells independent of the originating lineage of those cells. Further studies to evaluate the changes that occur in recurrent O-PDXs cells are underway.

Citation Format: Anand G. Patel, Elizabeth Stewart, Michael Clay, Xiang Chen, Michael Dyer. Single-cell characterization and lineage tracking of recurrent pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3768.
Abstract 3773: Investigating the tumor-host evolutionary arms race as a possible strategy for cancer therapy
Authors: Hashim Arig A. Ibrahim , Abrahams Dominique , Luddy Kim , Gillies Robert , Richards Christina L. , Brown Joel , Gatenby Robert
Published: August 2019

As first proposed by Cairns and Nowell more than 60 years ago, cancer is a Darwinian process so that cancer progression and resistance to therapy are governed by natural selection and eco-evolutionary dynamics. This creates an “evolutionary arms race” between the cancer cells and the tumor suppressor mechanisms of the host. To mechanistically investigate these dynamics we have conducted an experiment in which we selected for resistance to growth of implanted Lewis lung (mouse) cancer tumor (LL/2-Luc-M38) cells in immuno-deficient (SCID) mice and immuno-competent (C57BL/6). At each round a fixed numbers of LL/2-Luc-M38 cells were implanted in 10 male SCID and 10 male C57BL/6 mice. Tumor growth was measured and the two animals that exhibited the slowest tumor growth were bred and their male progeny were used as host in the subsequent round. After 12 generations, the tumors grew at approximately 1/10th the rate compared their initial generation. We observed that the hosts evolved cancer suppression through two general strategies. The evolved SCID mice suppressed tumor growth via mechanical restriction with increased in collagen deposition in and around the tumor. In contrast BL/6 mice enhance their immunologic response. We investigated the molecular properties of tumor cells growing in the different cohorts suing microarray. We identified genes significantly different in their expression of cancer cells harvested at different days following injection in Wild Type and Evolved SCID and BL/6 mice using Principle component analysis (PCA) method. The genes were functionally classified based on Gene Ontology and the majority was found to be related to integrin binding, cell adhesion molecular binding, and extra cellular matrix (ECM) binding in SCID mice with collagen, type XII, alpha (Col12a1) being the significantly decreased (results validated with RT-PCR (**p=0.009)). However, in BL/6 mice Molecular studies, contrary to expectation, did not show significant changes in expression of immune-related genes. Rather we found a decrease in expression of genes associated with the angiogenesis and ATP production as well as increased expression of genes regulating EMT. Thus, our results highly suggest that the counter adaptive strategies deployed by the cancer cells in response to the host suppressive mechanisms is host dependent and can be a possible target for therapy.

Citation Format: Arig A. Ibrahim Hashim, Dominique Abrahams, Kim Luddy, Robert Gillies, Christina L. Richards, Joel Brown, Robert Gatenby. Investigating the tumor-host evolutionary arms race as a possible strategy for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3773.
Abstract 3779: Clonal tracing reveals different mechanisms of resistance to immune checkpoint blockade
Authors: Gu Shengqing , Hu Xihao , Wang Xiaoqing , Jiang Peng , Li Ziyi , Traugh Nicole , Bu Xia , Xing Xiaofang , Freeman Gordon J. , Brown Myles , Liu Xiaole S.
Published: August 2019

Immune checkpoint blockade (ICB) therapy has improved patient survival in a variety of cancers, but its resistance presents a major clinical challenge. It remains unclear whether therapeutic resistance is driven predominantly by refractory clones of cancer cells or by immunosuppressive microenvironment. To address this, we utilized syngeneic transplantation model and performed clonal tracing of cancer cells to monitor the response to ICB. Different cancer clones displayed variable sensitivity to ICB treatment, reflecting intratumoral heterogeneity in therapy response. Furthermore, different recipients manifested distinct resistance mechanisms. ICB-responders developed resistance through selection and expansion of pre-existing ICB-resistant cancer clones, whereas non-responders presented with irresponsive microenvironment rather than dominance of resistant cancer clones. Extrapolation of the tumor-infiltrating immune repertoire revealed that higher IgG and lower IgA strongly correlated with better ICB response. This biomarker was further validated in multiple clinical cohorts. Our approach discriminates different sources of resistance to ICB, and identifies humoral immunity as biomarker of ICB response.

Citation Format: Shengqing Gu, Xihao Hu, Xiaoqing Wang, Peng Jiang, Ziyi Li, Nicole Traugh, Xia Bu, Xiaofang Xing, Gordon J. Freeman, Myles Brown, Xiaole S. Liu. Clonal tracing reveals different mechanisms of resistance to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3779.
Abstract 378: HDAC inhibition enhances MEK antagonist therapy in uveal melanoma through combined blockade of YAP, AKT and RTK signaling
Authors: Faiao-Flores Fernanda , Emmons Michael , Durante Michael , Saha Biswarup , Fang Bin , Koomen John , Chellappan Srikumar , Maria-Engler Silvya , Licht Jonathan , Harbour William , Smalley Keiran
Published: August 2019

Around 85-90% of all uveal melanomas harbor driver mutations in GNAQ or GNA11 leading to constitutive activation of numerous signaling pathways, including the MAPK pathway. MEK inhibitors have been evaluated clinically for metastatic uveal melanoma, but the responses are short-lived and the mechanisms of adaptation are poorly understood. In the current study, we performed RNA-seq and activity-based protein profiling (ABPP) to define the adaptive response of uveal melanoma cells to MEK inhibition and to design more effective combination therapy strategies. These analyses showed that MEK inhibition caused cytoskeleton remodeling driven by cortactin/Rho-GTPases with an increase in YAP activity, which in turn allowed therapeutic escape. Cortactin knockdown decreased YAP activity in response to MEK inhibition, increased cell death in vitro and was associated with tumor shrinkage in vivo. The proteomic data showed that MEK inhibition increased HDAC activity and an increase in global protein deacetylation. Co-targeting of HDACs and MEK was associated with increased apoptosis, decreased survival in 2D and 3D cell culture assays and suppression of YAP signaling. As YAP was unlikely to be the only escape pathway, we performed kinome and RTK arrays and demonstrated MEK inhibition also increases ROR1/2 and IGF-1R phosphorylation, leading to downstream PI3K/AKT signaling. At a signal transduction level, the combination of a pan-HDAC inhibitor (panobinostat) with a MEK inhibitor (trametinib) blocked all of the adaptive signaling pathways we identified, including RTKs, AKT, YAP and cortactin. In vivo xenograft studies revealed the MEK/HDAC inhibitor combination to outperform either agent alone, leading to long-term decrease of tumor growth and the suppression of adaptive PI3K/AKT, cortactin and YAP signaling. These findings identify HDAC inhibitors as a promising combination partner for MEK inhibitors in advanced uveal melanoma that may lead to improved systemic responses.

Citation Format: Fernanda Faiao-Flores, Michael Emmons, Michael Durante, Biswarup Saha, Bin Fang, John Koomen, Srikumar Chellappan, Silvya Maria-Engler, Jonathan Licht, William Harbour, Keiran Smalley. HDAC inhibition enhances MEK antagonist therapy in uveal melanoma through combined blockade of YAP, AKT and RTK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 378.
Abstract 3780: Spatially resolved transcriptional patterns of PARP inhibitor-resistant high grade serous ovarian cancer PDX tissue
Authors: King Benjamin , Oudenhove Elke Van , Misirlioglu Selim , Fernandez Ernesto Arostegui , Levine Douglas , Lionnet Timothee
Published: August 2019

Ovarian cancer is the leading cause of mortality among gynecological malignancies; 70% of patients who initially respond to therapy eventually relapse and die. Within highly heterogeneous tumors, resistance originates from a small number of cells that express beneficial genes and receive favorable cues from their microenvironment. Understanding the mechanisms of drug resistance therefore requires the knowledge of which cell types compose the tumor, which genes they express, and where they are located respective to other cells and tissue landmarks. To this end, we have explored cell type and transcriptional heterogeneity in homologous recombination deficient patient-derived xenograft (PDX) models of high grade serous ovarian cancer (HGSOC) treated with the PARP inhibitor talazoparib. We are using single-cell RNA sequencing (scRNA-seq) to identify the discrete cell types present in drug naïve and drug resistant PDX tissue and to characterize the transcriptional changes driving the resistant phenotype. We are using single-molecule hybridization chain reaction (smHCR) to fluorescently label mRNAs and diSPIM light sheet microscopy to visualize fluorescently labeled mRNAs within large volumes of PDX tissue. We are currently developing computational strategies to deconvolve the 3D spatial organization of PDX cell types and to understand where and when resistance genes are expressed. This integrated scRNA seq and smHCR microscopy framework has allowed us to identify rare cell populations within HGSOC PDXs and to visualize their 3D localization relative to other cell types and tissue features, and it has the potential to elucidate how transcription of key genes supports transition to a resistant state. The insights gained from single cell transcriptional and 3D spatial profiles has the potential to greatly advance our understanding of how the 3D tumor tissue microenvironment allows and encourages rare cells with pre-resistant transcriptional programs to escape PARP inhibitor treatment. Further, this research has the potential to be predictive of disease progression and to suggest additional therapeutic targets that could be exploited as part of novel and/or personalized combination therapy regimens. An additional advantage is that this approach could be generalizable to many types of cancer to characterize the interplay between tissue organization and diverse processes such as tumor evolution, metastasis, and drug resistance.

Citation Format: Benjamin King, Elke Van Oudenhove, Selim Misirlioglu, Ernesto Arostegui Fernandez, Douglas Levine, Timothee Lionnet. Spatially resolved transcriptional patterns of PARP inhibitor-resistant high grade serous ovarian cancer PDX tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3780.
Abstract 3788: Sensitive detection of oncoviruses integrated into a comprehensive tumor immuno-genomics platform
Authors: Bartha Gabor , Li Robin , Luo Shujun , West John , Chen Richard
Published: August 2019

Introduction

HPV, HBV, HCV and EBV viruses are causally linked to over 11% of cancers worldwide while KSHV, HTLV and MCV are linked to an additional 1%. As use of immunotherapy expands to a broader variety of cancers, it is important to understand how these oncoviruses may be impacting the overall immune response in patients as part of the tumor and tumor microenvironment. However, typical cancer diagnostic and genomic biomarker assays do not include oncovirus detection.

Methods

To enable detection of these oncoviruses, we have developed ImmunoID NeXT, a novel augmented exome and transcriptome based platform that comprehensively characterizes tumor and tumor microenvironment from a single sample. As part of the platform assay design we developed targeting for the following oncoviruses: HPV, HBV, HCV, EBV, KSHV and HTLV. Furthermore we developed analytics for detecting these viruses from both the DNA and RNA data. To test the ability of the platform to detect these oncoviruses, we identified a set of 11 EBV cell lines from Coriell in which EBV was used as a transformant. We obtained another 23 cell lines from ATCC containing HPV16, HPV18, HPV45, HPV68, HBV, EBV, KSHV, HTLV1 and HTLV2 in which the oncoviruses were known to be in the tumors from which the cell lines were created. We have also started running a series a FFPE tumor samples of unknown status. We extracted DNA and RNA from all these samples, made libraries and sequenced them on a NovaSeq to 30G. High quality read counts were computed for all targeted oncoviruses.

Results

We detected EBV in all the Coriell cell lines in both DNA and RNA indicating strong sensitivity of the platform. Wide dynamic range suggests quantification may be possible as well. In the DNA there was no signal from any other oncovirus indicating high specificity. In the ATCC samples we detected 23 out of 23 oncoviruses expected in both the DNA and RNA. We detected all the different types of oncoviruses that we targeted except for HCV because it wasn’t in any sample. In all but one case the signals were strong. False positive detections appear to be rare. Much less than 1% of the overall exome/transcriptome reads map to oncoviruses in all cases.

Conclusions

Oncovirus detection integrated into an augmented exome and transcriptome assay is sensitive and specific. Oncoviral characterization of the tumor as part of a comprehensive tumor immunogenomics platform can serve and an important additional biomarker for understanding immunotherapy response.

Citation Format: Gabor Bartha, Robin Li, Shujun Luo, John West, Richard Chen. Sensitive detection of oncoviruses integrated into a comprehensive tumor immuno-genomics platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3788.
Abstract 3790: Molecular analysis of HPV genotype in head and neck cancers in a tertiary hospital in Ghana
Authors: Duduyemi Babatunde M. , Yeboah Francis A. , Aboagye Evans
Published: August 2019

Introduction: Human papillomavirus (HPV) has been implicated in head and neck cancers (HNC), and patients now present at a much younger age without a strong alcohol or tobacco history. This work aims to establish the prevalence of HPV in head and neck squamous cell carcinoma (HNSCC) in our center as very minimal work has been done in Ghana in this area.

Method: The study was to detect HPV DNA and genotypes from histologically confirmed HNSCC. Formalin-fixed paraffin embedded (FFPE) tissue blocks and slides of HNSCC cases were retrieved consecutively. Genomic DNA was then prepared from 10μm FFPE tissue section using the Quick-DNATM FFPE kit. A nested multiplex PCR assay that combines degenerate E6/E7 consensus primers and type-specific primer was utilized for the detection of HPV genotypes (16, 18, 31, 33, 35, 45, 51, 52, 56, 58, 59, 6/11, 42, 43 and 44) in three cocktails.

Results: A total of 301 HNSCC cases were extracted from the surgical day book, with male preponderance of 72.1% and a mean age of 54.97 years. The most common site of occurrence was the oral cavity (28.9%), followed by larynx (26.2%). Majority of the samples of patients aged <30 years were poorly differentiated (65.7%). HPV E6/E7 oncogenic DNA was detected in 18% (n=100), with 17 being HPV-16 and 1 both HPV-16 and HPV-18. A higher prevalence of HPV-positive DNA was found in males (83.3%), and there was a trend towards patients older than 56 years likely to have HPV tumours (OR=1.378). Larynx was the commonest site for HPV-positive HNSCC (38.9%), followed by oropharyngeal site (33.3%).

Conclusion: The overall prevalence of HPV DNA (genotypes 16 and 18) in our study was 18%, more common in younger people and mainly male. This suggests that males should be considered in screening and vaccination against HPV to prevent HNSCC.

Citation Format: Babatunde M. Duduyemi, Francis A. Yeboah, Evans Aboagye. Molecular analysis of HPV genotype in head and neck cancers in a tertiary hospital in Ghana [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3790.
Abstract 380: Tumor suppressor retinoblastoma status as a predictor of ATR inhibitor sensitivity
Authors: Mancusi Caterina , Hartley John , Mittnacht Sibylle
Published: August 2019

Inhibitors of the Ataxia Telangiectasia and Rad3 related protein kinase (ATR) are increasingly entering clinical trials as single agents or in combination with inhibitors of poly (ADP-ribose) polymerases (PARPs), DNA-damaging chemotherapy or radiotherapy. However, and despite promise of broad utility and cancer selectivity, compelling results have remained an exception, underscoring the need to identify responsive patient population and tumour types. Based on in vitro testing in an extended cancer cell line panel we found that deleterious mutation of RB1 is significantly associated with hypersensitivity to multiple clinically relevant ATR inhibitors. Cancer cell lines with deleterious RB1 mutation displayed significantly enhanced cell death following ATR inhibitor treatment compared to a histotype matched cancer cell line groups with RB1 normal status and depletion of RB1 gene in RB1 normal cancer cell lines using siRNA, increased sensitivity to ATR inhibition accompanied by increased cells death. Deletion or deleterious mutation of RB1 is frequent in small cell lung cancers, cancers of neuroendocrine origin, and a considerable portion of triple negative breast and urinary tract cancers, cancers of the nervous system, sarcoma and chronic lymphatic leukemia. The data obtained document a link between RB1 status and ATR inhibitor sensitivity and argue that focus on cancer types with RB1 mutation or 13q14 involvement, where RB1 is encoded, should be considered in the design and analysis of trials involving ATR inhibitors.

Citation Format: Caterina Mancusi, John Hartley, Sibylle Mittnacht. Tumor suppressor retinoblastoma status as a predictor of ATR inhibitor sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 380.
Abstract 3802: Interleukin-4 receptor-targeted delivery of Bcl-xL siRNA sensitizes tumors to chemotherapy and inhibits tumor growth
Authors: Lee Soo-Woong , Yoon Jae-Won Yoon , Padmanaban Guruprasath , Park Rang- Woon , Baek Moon-Chang , Kim Dong-Kyu , Kim Won-Jong , Lee Byungheon
Published: August 2019

IL-4 receptor (IL-4R) is commonly up-regulated on tumor cells, and interactions between the receptor and Interleukin-4 (IL-4) can induce the expression of anti-apoptotic proteins, including Bcl-xL. This contributes to tumor cell survival and their resistance to chemotherapy. In this study, we exploited IL-4R-targeted delivery of Bcl-xL siRNA to IL-4R-expressing tumor cells in order to sensitize them to chemotherapy. To target IL-4R, an IL-4R-binding peptide, IL4RPep-1, was attached to branched polyethyleneimine-superparamagnetic iron oxide nanoparticles (BPEI-SPION). These nanoparticles were then complexed with Bcl-xL-targeting siRNA. IL-4R-targeted BPEI-SPION/Bcl-xL siRNA more efficiently reduced Bcl-xL gene expression and enhanced cytotoxicity of doxorubicin in MDA-MB231 breast tumor cells compared to untargeted BPEI-SPION/Bcl-xL siRNA. The siRNA was released from the complexes after 15 h of incubation at pH 5.5 and was stable in the complexes up to 72 h in the serum. The IL-4R-targeted BPEI-SPION/siRNA was internalized by cells through IL-4R, successfully escaped the endosomes, and was dispersed into the cytoplasm. Near-infrared fluorescence and magnetic resonance imaging demonstrated that in vivo tumor homing and accumulation of IL-4R-targeted BPEI-SPION/siRNA were both higher than untargeted BPEI-SPION/siRNA. The IL-4R-targeted BPEI-SPION/Bcl-xL siRNA, in combination with doxorubicin, significantly inhibited tumor growth in mice compared to untargeted BPEI-SPION/Bcl-xL siRNA. These results suggest that the IL-4R-targeted delivery of Bcl-xL siRNA to IL-4R-expressing tumors can sensitize tumors to chemotherapy and enhance the efficacy of anti-tumor therapeutics. Keywords: Bcl-xL, BPEI-SPION, chemotherapy, IL-4R, siRNA

Citation Format: Soo-Woong Lee, Jae-Won Yoon Yoon, Guruprasath Padmanaban, Rang- Woon Park, Moon-Chang Baek, Dong-Kyu Kim, Won-Jong Kim, Byungheon Lee. Interleukin-4 receptor-targeted delivery of Bcl-xL siRNA sensitizes tumors to chemotherapy and inhibits tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3802.
Abstract 3803: Targeting 17q23 amplicon to overcome the resistance to anti-HER2 therapy in HER2+ breast cancer
Authors: Liu Yunhua , He Xiaoming , Lu Xiongbin , Zhang Xinna
Published: August 2019

Amplification of chromosome 17q23 is a frequent genomic event that occurs in ~ 11% of human breast cancers. The 17q23 amplification is enriched in HER2+ breast cancers, which is significantly correlated with poor clinical outcomes. Previous studies have identified the oncogenic phosphatase WIP1 gene in the amplicon, which functions as a master inhibitor in DNA damage response. While the possibility of any other protein-coding oncogenes in the WIP1-containing 17q23 amplicon was ruled out, our analysis of human breast cancer genomics uncovered an oncogenic microRNA gene, MIR21, in a majority of the WIP1-containing amplicons. Accordingly, the 17q23 amplification results in aberrant expression of WIP1 and miR-21, which not only promotes breast tumorigenesis, but also leads to resistance to anti-HER2 therapies. Inhibiting WIP1 and miR-21 using small molecular inhibitor against WIP1 (GSK2830371) and anti-miR-21 oligonucleotides selectively inhibits the proliferation, survival and tumorigenic potential of HER2+ breast cancer cells harboring 17q23 amplification. However, the in vivo bioavailability of the two agents in their free form is poor. To overcome the resistance of trastuzumab-based therapies in vivo, we developed pH-sensitive nanoparticles for specific co-delivery of the two agents into breast tumors. The nanoparticles can improve the encapsulation efficiency of anti-miR-21 oligonucleotides but also effectively capture carbon dioxide (CO2) into the nanoparticle to achieve the ‘nano-bomb’ effect for triggered drug release under the reduced pH in tumors. The two agents (inhibitors of miR-21 and WIP1)-laden nanoparticles can be used to efficiently kill trastuzumab-resistant HER2+ breast cancer cells, leading to a profound reduction of the tumor growth in vivo. These results demonstrate the great potential of the combined treatment of WIP1 and miR-21 inhibitors for the HER2+ breast cancers resistant to anti-HER2-based therapies.

Citation Format: Yunhua Liu, Xiaoming He, Xiongbin Lu, Xinna Zhang. Targeting 17q23 amplicon to overcome the resistance to anti-HER2 therapy in HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3803.
Abstract 3806: Overcoming drug-tolerant cancer cell subpopulations showing AXL activation and epithelial-mesenchymal transition is critical in conquering ALK-positive lung cancer
Authors: Nakamichi Shinji , Masahiro Seike , Miyanaga Akihiko , Takahashi Akiko , Noro Rintaro , Kubota Kaoru , Gemma Akihiko
Published: August 2019

Anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKIs) induce a dramatic response in non-small cell lung cancer (NSCLC) patients with the ALK fusion gene. However, acquired resistance to ALK-TKIs remains an inevitable problem. In this study, we aimed to discover novel therapeutic targets to conquer ALK-positive lung cancer. We established three types of ALK-TKI (crizotinib, alectinib and ceritinib) -resistant H2228 NSCLC cell lines by high exposure and stepwise methods. We found these cells showed a loss of ALK signaling, overexpressed AXL with epithelial-mesenchymal transition (EMT), and had cancer stem cell-like (CSC) properties, suggesting drug-tolerant cancer cell subpopulations. Similarly, we demonstrated that TGF-β1 treated H2228 cells also showed AXL overexpression with EMT features and ALK-TKI resistance. The AXL inhibitor, R428, or HSP90 inhibitor, ganetespib, were effective in reversing ALK-TKI resistance and EMT changes in both ALK-TKI-resistant and TGF-β1-exposed H2228 cells. Tumor volumes of xenograft mice implanted with established H2228-ceritinib-resistant (H2228-CER) cells were significantly reduced after treatment with ganetespib, or ganetespib in combination with ceritinib. Some ALK-positive NSCLC patients with AXL overexpression showed a poorer response to crizotinib therapy than patients with a low expression of AXL. ALK signaling-independent AXL overexpressed in drug-tolerant cancer cell subpopulations with EMT and CSC features may be commonly involved commonly involved in intrinsic and acquired resistance to ALK-TKIs. This suggests AXL and HSP90 inhibitors may be promising therapeutic drugs to overcome drug-tolerant cancer cell subpopulations in ALK-positive NSCLC patients for the reason that ALK-positive NSCLC cells do not live through ALK-TKI therapy.

Citation Format: Shinji Nakamichi, Seike Masahiro, Akihiko Miyanaga, Akiko Takahashi, Rintaro Noro, Kaoru Kubota, Akihiko Gemma. Overcoming drug-tolerant cancer cell subpopulations showing AXL activation and epithelial-mesenchymal transition is critical in conquering ALK-positive lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3806.
Abstract 3816: Corilagin could help to overcome PARP inhibitor resistance by inhibiting ERK signaling pathways
Authors: Luan Baoxin , Zhao Hongbo , Bast Robert C. , Lu Zhen , Yu Yinhua
Published: August 2019

We have identified that Corilagin is a major anti-tumor active component extracted from a well-known hepatoprotective and antiviral medicinal herb, Phyllanthus niruri L. Our previous work found that Corilagin inhibited the growth of ovarian cancer cells via TGF-β/AKT/ERK signaling pathways. Corilagin also sensitizes Paclitaxel and Carboplatin by primarily inhibiting Snail-glycolysis pathways. Recently, we have investigated if Corilagin could give a help to overcome poly ADP ribose polymerase (PARP) inhibitors (PARPi) resistance in ovarian cancer cells. As we knew, PARP inhibitors are DNA repair drugs, have been approved for ovarian cancer therapy, but drug resistance is a faced problem, combination therapy may give helps. In this study, two pairs of PARPi sensitive/resistant cells: A2780CP/A2780CP_R and UMB1.289/UMB1.289_R (Sci Transl Med. 2017; 9:392) were used. Cells were cloned again and confirmed their sensitivities to PARPi. The IC50 of PARPi-BMN673 is 0.02μM to A2780CP, > 10μM to A2780CP_R, 0.12μM to UMB1.289 and 60μM to UMB1.289_R. The combination assays showed Corilagin had almost equally cytotoxic activities to these two pairs and had synergistic effects with BMN673 to A2780CP_R and UMB1.289_R . Using CulcuSyn, the combination indexes (CI) presented at IC50 are 0.17365 and 0.62787 in A2780_R and UMB1.289_R, respectively. Signaling analysis found that PARPi did down-regulate the expression levels of PARP and up-regulate of pH2AX, PARPi only inhibited pERK activity in sensitive cells, but not in resistant cells. In contrast, Corilagin did not have DNA repair function, but it inhibited pERK activity in both sensitive and resistant cells. In summary, we demonstrate that combined treatment with Corilagin and PARPi evokes synergistic cytotoxic effects in two pairs of ovarian cancer cell models. Corilagin is an herb medicine with lower toxic effects to normal cells, especially hepatoprotective, which could be an ideal complimentary medicine when combinating with PARPi in ovarian cancer therapy.

Citation Format: Baoxin Luan, Hongbo Zhao, Robert C. Bast, Jr., Zhen Lu, Yinhua Yu. Corilagin could help to overcome PARP inhibitor resistance by inhibiting ERK signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3816.
Abstract 3823: Inhibition of homologous recombination, PARP inhibitor, or dianhydrodulcitol overcomes temozolomide-resistance in glioma cells
Authors: OHBA SHIGEO , Hirose Yuichi
Published: August 2019

Glioblastoma is one of the most aggressive tumors in the central nervous system tumors. The standard therapy for glioblastomas is maximal safe resection, followed by radiation therapy and chemotherapy with temozolomide (TMZ). The acquisition of resistance to TMZ is big problem. TMZ is a DNA-methylating agent, delivering a methyl group to DNA (O6-guanine, N7-guanine and N3-adenine). The primary cytotoxic lesion, O6-methylguanine, mispairs with thymine, leading to futile DNA mismatch repair (MMR), formation of double strand breaks and eventual cell death, when O6-methylguanine DNA methyltransferase (MGMT) is absent. N7-methylguanine and N3-methyladenine are repaired by base excision repair. The object of the study was to reveal the mechanisms of resistance to TMZ and to find the way to overcome the resistance in glioma cells. Several clones of TMZ-resistant U251 were obtained and analyzed. #3 clone showed G2 arrest after TMZ exposure and this arrest was abrogated sooner compared to parental U251. TMZ did not induce G2 arrest in #8 clone. The expression of MGMT was not found in U251 parental cells, #3 cells nor #8 cells. The ability of homologous recombination (HR) was increased in #3 clone, and suppression of HR resensitized #3 clone to TMZ. The protein levels of MSH6, which was one of the main proteins associated with MMR, was reduced in #8 clone. Inhibition of base excision repair by PARP inhibitor resensitized #8 clone to TMZ. Dianhydrodulcitol, alkylating agent, suppressed the proliferation in U251 and even U251-derived resistant clones. These effects were shown in U87MG-dereived resistant clones and MGMT-proficient cell lines. To select the way according to the mechanisms of resistance to TMZ could overcome the resistance in glioma cells.

Citation Format: SHIGEO OHBA, Yuichi Hirose. Inhibition of homologous recombination, PARP inhibitor, or dianhydrodulcitol overcomes temozolomide-resistance in glioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3823.
Abstract 3856: MTLCEBPA, a drug candidate for hepatocellular-carcinoma enhances efficacy of Sorafenib
Authors: Habib Nagy A. , Huang Kai-Wen , Reebye Vikash , Sodergren Mikael , Rossi John
Published: August 2019

MTLCEBPA, a non-toxic therapeutic oligonucleotide formulated inside SMARTICLES (liposomal nanoparticles) is currently under a Phase I study for patients with advanced hepatocellular carcinoma (HCC). We have demonstrated in multiple in vivo liver disease models that MTLCEBPA can improve liver function and inhibit HCC tumor growth. Here we show that upregulation of hepatic CEBPA expression by MTLCEBPA enhances the effects of Sorafenib in a (Diethylnitrosamine) DEN induced cirrhotic HCC rat model by significantly reducing liver tumor volume after only two weeks of combination treatment when compared to untreated control or single agent treatment only.

In this study, we established cirrhosis and HCC by exposing male Wistar rats to DEN for 6 weeks. Animals were randomized into 5 groups comprising of:

1. PBS control;

2. MTL-CEBPA treatment (TIW at 3mg/kg) for one week;

3. MTL-CEBPA treatment (TIW at 3mg/kg) for two weeks;

4. Sorafenib TIW at 10mg/kg for two weeks; and

5. MTLCEBPA at 3mg/kg at week one combined with Sorafenib 10mg/kg at week 2 group.

The results here indicate that Sorafenib combined with MTLCEBPA significantly inhibits tumor growth when compared to single agent treatment. Tumor sizes averaged at 644.7mm3 (± 88.57) in group 1 compared to 326mm3 (± 53.05) in group 2; 199.7mm3 (± 54.73) in group 3 and 299.5mm3 (± 77.1) in group 4. In combination treatment (group 5) the tumor

sizes averaged at 101.3mm3 (± 37.48). Serum AFP change (posttreatment values - pre-treatment) was more prominent in the MTLCEBPA/Sorafenib combination treatment (group 5) where there was a 4-fold reduction in values after treatment when compared to control groups 1-4. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and total bilirubin remained unchanged across all the treatment groups suggesting no deterioration in liver function during the study period.

These results suggest that MTLCEBPA improved the efficacy of Sorafenib in this DEN induced HCC model, indicating a promising therapeutic option for advanced HCC patients who can no longer be treated with potentially more effective local therapies.

Citation Format: Nagy A. Habib, Kai-Wen Huang, Vikash Reebye, Mikael Sodergren, John Rossi. MTLCEBPA, a drug candidate for hepatocellular-carcinoma enhances efficacy of Sorafenib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3856.
Abstract 3872: Targeting multi-functional ERK-protein complexes in vivo
Authors: Kaoud Tamer S. , Johnson William H. , Ebelt Nancy D. , Piserchio Andrea , Zamora-Olivares Diana , Ravenstein Sabrina V. , Pridgen Jacey R. , Edupuganti Ramakrishna , Sammons Rachel , Cano Micael , Warthaka Mangalika , Ren Pengyu , Anslyn Eric V. , Tsai Kenneth Y. , Ghose Ranajeet , Dalby Kevin N.
Published: August 2019

Mutations in pathways that enhance the activity of ERK1 and ERK2 are frequently present in human cancers, reflecting their important roles in tumor initiation, and progression. This notion is reinforced by observations in BRAF V600E mutant melanoma where the majority of the mechanisms of resistance to FDA-approved combination therapies targeting BRAF and MEK involve reactivation of ERK1 and ERK2. Recently, the direct targeting of the ERK enzymes using ATP-competitive inhibitors has emerged as an attractive strategy to overcome acquired resistance to current combination therapies. The ERK enzymes employ unique mechanisms of molecular recognition to engage protein components of the MAPK pathway. Here we report the potent targeting of an ERK-protein docking interaction by a small molecule thiotriazole, which abrogates ERK signaling in vivo. The thiotriazole binds covalently to a highly conserved cysteine residue within the D-recruitment site of ERK1/2 with more than a 100-fold discrimination over other MAPKs (e.g. JNK1/2, p38MAPKs and ERK5). Treatment of various BRAF-inhibitor naive or inhibitor-resistant melanoma cell lines expressing BRAF V600E with the thiotriazole for 2 hours induces dose-dependent inhibition of ERK activation and downstream signaling. Inhibition is maintained for up to 5 hours following thiotriazole washout and induces apoptosis and growth inhibition. Treatment of mice carrying a BRAFV600E A375 melanoma xenograft with the thiotriazole blocked tumor growth. Transient expression of a mutant form of ERK2, which is resistant to the thiotriazole, promotes survival of A375 and HEK 293 cells treated with thiotriazole. This study lays the foundation for developing a new modality for the treatment of solid tumors driven by excessive ERK signaling.

Citation Format: Tamer S. Kaoud, William H. Johnson, Nancy D. Ebelt, Andrea Piserchio, Diana Zamora-Olivares, Sabrina V. Ravenstein, Jacey R. Pridgen, Ramakrishna Edupuganti, Rachel Sammons, Micael Cano, Mangalika Warthaka, Pengyu Ren, Eric V. Anslyn, Kenneth Y. Tsai, Ranajeet Ghose, Kevin N. Dalby. Targeting multi-functional ERK-protein complexes in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3872.
Abstract 3876: Thymoquinone induces the expression of clock genes in breast cancer cells
Authors: Alamoudi Aliaa A. , Bashmail Hanan A. , Ajabnoor Ghada M. , Choudhry Hani , Hassan Mohammed A. , Aldahlawi Alia M. , Al-abd Ahmed A.
Published: August 2019

Thymoquinone is naturally occurring bioactive compound which showed cumulative preclinical evidence of anticancer effects. However, its exact anti-cancer activity remains to be elusive and is currently being studied. In this study, we assessed the role of thymoquinone as a potential therapy in human breast cancer cell lines in addition to its role in altering molecular clock genes expression, which in turn has been associated with tumorgenesis. Sulfa-Rhodamine-B assay (SRB) assay was used to evaluate the cytotoxic effect of thymoquinone in breast cancer cell lines (MCF-7 and T47D). Following 72 h of exposure, thymoquinone showed cytotoxic effects against MCF-7 and T47D with IC50’s of 44.4±3 µM and 152±11 µM respectively. Further investigation showed that thymoquinone showed some affect on the cell cycle distribution in both cell lines. It significantly increased the cell population in S-phase in MCF-7cells (P<0.05), while causing a significant G1 phase arrest in T47D cells (p<0.01). Interestingly, treatment of both cell lines with non-cytotoxic concentrations of thymoquinone resulted in a significant increased expression of clock genes, measured by reverse transcription–quantitative PCR (RT-qPCR). Thymoquinone appeared to increase the expression of BMAL-1 (1.4±0.1 fold), CLOCK (3.4±0.9 fold), PER-1(5.6±0.5 fold), CRY-1 (4.4±0.4 fold), CRY-2(2.2±0.6 fold) in MCF-7 cells. Similarly, thymoquinone increased the expression of BMAL-1 (1.4±0.3 fold), CLOCK (1.4±0.3 fold), PER-1(1.4±0.3 fold), CRY-1 (1.5±0.4 fold), and CRY-2 (1.8±0.1 fold) in T47D cells . In conclusion, thymoquinone possesses a potential in combating breast cancer alone. Despite the promising anti-proliferative activity of thymoquinone against breast cancer cells, the better understanding of these effects is needed to provide a novel approach for the treatment of breast cancer as a disease. Here, we postulate that the drug could be modulating clock genes expression, which ultimately can affect key molecules in cell cycle and cell proliferation. Further studies are needed to confirm this and to elucidate the exact underlying mechanism.

Citation Format: Aliaa A. Alamoudi, Hanan A. Bashmail, Ghada M. Ajabnoor, Hani Choudhry, Mohammed A. Hassan, Alia M. Aldahlawi, Ahmed A. Al-abd. Thymoquinone induces the expression of clock genes in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3876.