bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2796: Validation of Brightplex, a multiplexed IHC solution for immune cell phenotyping of the tumor microenvironment
Authors: Collignon Aurélie , Benchaaben Assil , Kassambara Alboukadel , Guimaraes Felipe , Prestat Emmanuel , Haond Christophe , Fieschi Jacques
Published: August 2019

Brightplex is a multiplexing solution developed by HalioDx to analyze the tumor microenvironment on FFPE tissue. It leverages chromogenic immunohistochemistry performed by iterative automated immunostaining on the same slide followed by whole slide digital pathology analysis based on proprietary software combined to Halo from Indica Lab. Stained cells can be quantified in various regions of interest (tumor, stroma, invasion margin). The Brightplex T cell exhaustion (TCE) panel includes 5 biomarkers (CD3, CD8, PD1, TIM3 and LAG3) to assess the expression of three immune checkpoints on T cells. This panel is intended to be used in clinical research to identify patients susceptible to respond to new immunotherapies especially in the context of the resistance to anti PD(L)1 treatment. Therefore, it is critical to thoroughly demonstrate the robustness and the accuracy of such complex assay. For the validation study of the TCE panel, tissue sections were sequentially immunostained for TIM3, PD1, LAG3, CD8 and CD3. Following each immunostaining, the slides were digitized, de-stained and the antibodies stripped before the next immunostaining. Images of the whole slide were then coregistered and analyzed by digital pathology to determine the densities of various T cell populations: CD8+ T lymphocytes expressing of 1, 2 or 3 immune checkpoints. The precision of the assay was evaluated in terms of intra- and inter-run repeatability and the accuracy was evaluated by comparison of the automated multiplex assay with singleplex staining. Results demonstrate the reliability of Brightplex to analyze tumor sections. The precision study showed that Brightplex was reproductible on several tumor types such as NSCLC, colon and breast and the accuracy was demonstrated by comparison of cell density for each biomarker following either multiplex or singleplex staining. The high degree of robustness could be explained notably by the iterative chromogenic immunohistochemistry technology which limits the risks of steric hindrance when antibodies and amplification systems are used simultaneously and by the intrinsic robustness of this routine workflow including IVD grade instrument and reagents.

Citation Format: Aurélie Collignon, Assil Benchaaben, Alboukadel Kassambara, Felipe Guimaraes, Emmanuel Prestat, Christophe Haond, Jacques Fieschi. Validation of Brightplex, a multiplexed IHC solution for immune cell phenotyping of the tumor microenvironment [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 2796.
Abstract 2797: Accumulation of neutrophils in the tumor microenvironment promotes resistance to immunotherapy in pancreatic cancer
Authors: Siolas Despina , Vucic Emily , Cullis Jane , Choi Mark , Bar-Sagi Dafna
Published: August 2019

The prognosis for pancreatic cancer patients is unequivocally poor. This is due in part to the fibrotic and immunosuppressive microenvironment of pancreatic tumors, which serves as a physical barrier to chemotherapy and immunotherapy. Intratumoral genetic alterations can cause remodeling of the immune environment through distinct cellular mechanisms. Oncogenic Kras gene mutations are the most frequently occurring genetic event in pancreatic cancer, followed by transforming alterations in TP53. We show that CRISPR/Cas engineered KrasG12D;Trp53R172H pancreatic ductal tumors have a distinct immune profile characterized by an increase in neutrophils and a concomitant decrease in cytotoxic and Th1+ T cells. In addition, KrasG12D;Trp53R172H pancreatic ductal tumors have an increase in CXCL5, a chemokine involved in neutrophil accumulation. Analysis of publicly available datasets shows that increased neutrophil numbers significantly correlate with decreased survival in pancreatic cancer patients. Of clinical relevance, p53-Crispr/Cas engineered pancreatic tumors in mice are resistant to immunotherapy/chemotherapy combination treatment. This data supports the possibility of personalizing immunotherapy treatments based on immune profiles.

Citation Format: Despina Siolas, Emily Vucic, Jane Cullis, Mark Choi, Dafna Bar-Sagi. Accumulation of neutrophils in the tumor microenvironment promotes resistance to immunotherapy in pancreatic 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 2797.
Abstract 2831: Detection of methane-producing archaea in the cervix of Hispanics using shotgun metagenomics
Authors: Godoy-Vitorino Filipa , Vazquez-Sanchez Frances , Dominicci-Maura Anelisse , Romaguera Josefina
Published: August 2019

The human cervix is colonized by microbial communities that have traditionally been regarded as a protective barrier against infections. Dysbioses disrupting the balance of the normal microbiota, leads to the appearance of anaerobic taxa that have been related to sexual transmitted infections and cancer. The complexity of the diversity and composition of cervicovaginal communities has been recently highlighted by 16S rDNA surveys, although these have limitations to either select mostly bacteria and the lack of strong lower taxonomic resolution such as being species-specific. Metagenomic shotgun sequencing overcomes these limitations, and gives us access to the extent of diversity beyond bacterial communities. This is the first project to characterize the bacterial and archaeal communities of the cervix of Puerto Ricans using shotgun metagenomics. Women coming for gynecology and colposcopy evaluation at the UPR and San Juan City clinics (San Juan Metropolitan area), and who did not meet several excluding criteria including having taken antibiotics for the last month, were recruited for the study. Samples were acquired by inserting a speculum for access and visualization of the cervix and a sterile swab was rotated along the lumen with a circular motion and frozen immediately for posterior genomic DNA extractions and sequencing. Library construction and shotgun sequencing of 10 cervical patient samples was done with the Illumina HiSeq platform An average of 1,690,000 good quality reads per sample were analyzed. Approximately 90% of the contigs had more than 1,000bp and 10% had sizes ranging from 1,000-5,000bp. Microbial communities were dominated by Lactobacillus, including L. iners, Gardnerella vaginallis, Nocardia and Atapobium vaginae. Archaeal diversity was dominated by Methanosarcina (namely, M. barkeri) with other less dominant taxa such as Methanolobus, Methanococcus, Natrococcus and Methanobrevibacter. The dominance of Lactobacillus was expected, and as Archaea are environmental organisms that have been associated with the mucosa in mammals, including protecting the human gut, they must have an important role in the cervix. As methanogenic archaea are known to remove fermentation end products, such as methanol and ethanol, as end-products of other fermentative bacteria, methanogenesis maybe important in preventing the accumulation of gases and other reaction end products that could damage the cervical mucosa.

Citation Format: Filipa Godoy-Vitorino, Frances Vazquez-Sanchez, Anelisse Dominicci-Maura, Josefina Romaguera. Detection of methane-producing archaea in the cervix of Hispanics using shotgun metagenomics [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 2831.
Abstract 2839: Leveraging gut microbiota networks to impact tumor immunotherapy
Authors: Jayaraman Lata , Sceneay Jaclyn
Published: August 2019

The human gut microbiota forms a diverse, dynamic and complex ecosystem that modulates numerous host processes including metabolism, inflammation and cellular and humoral immune responses. Emerging data suggest that the gut microbiota of cancer patients may predict tumor response to immune checkpoint inhibitors (ICI). To better understand how the microbiome may impact response to ICI, we have developed and validated robust tumor models using either conventional mice treated with antibiotics or germ-free (GF) mice. We have confirmed in both models, that mice lacking a diverse microbiome fail to mount an efficient anti-tumor immune response after treatment with anti-PD-1, primarily due to T cell dysfunction. This response to anti-PD-1 can be restored by introduction of a microbiome using fecal microbial transplant (FMT) prepared from stool from healthy donors in GF mice, and is driven by increased tumor-infiltrating lymphocytes (TILs), specifically CD8+ T cells. Importantly, for the first time, we show that the bacterial spore fraction from healthy donor stool can also restore response to anti-PD-1 and increase CD8+ TILs in both conventional mice treated with antibiotics and GF mice. We have also tested cancer patient-derived microbiome samples that are efficacious or non-efficacious in GF mouse models. We have sequenced the input material as well as the engrafted gut microbiota in these mice via 16S rDNA sequencing. Comparison of these sequences has enabled us to develop a “microbiome signature” of response that is being used in the development of a microbiome-based therapy for combination with ICI. Based on these encouraging animal model data we plan to initiate a randomized, placebo-controlled clinical study in patients with advanced metastatic melanoma. The clinical trial will evaluate the impact of an anti-PD-1 checkpoint inhibitor with adjunctive microbiome therapy on patient outcomes. Seres is developing SER-401, a preclinical stage oral microbiome therapy to improve the efficacy and safety of immunotherapy. Our drug discovery strategy combines computational analyses and empirical in vitro, in vivo and ex-vivo screening of strains and consortia to inform selection and drive microbiome drug design. Data from such a comprehensive approach are invaluable for designing compositions of bacteria that form “functional ecological networks” that can impact response to ICI therapy. We believe these data will provide insight into how microbiome drugs can be developed in the setting of immunotherapy to augment the efficacy of ICIs by altering the cancer-immune set point.

Citation Format: Lata Jayaraman, Jaclyn Sceneay. Leveraging gut microbiota networks to impact tumor immunotherapy [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 2839.
Abstract 2842: Vesal Yaghoobi
Authors: Yaghoobi Vesal , Pelekanou Vasiliki , O'Meara Tess , Silber Andrea , Pusztai Lajos , Rimm David
Published: August 2019

Background: It has been shown that even after adjusting for clinical stage at presentation and socio-economic variables, Triple Negative Breast Cancer (TNBC) still has a worse overall outcome in African American (AA) compared to Non-African American (NAA) patients. The abundance and composition of immune cells in the tumor microenvironment is a powerful prognostic factor in TNBC. Our goal was to assess if differences exist in CD8, CD68 and PD-L1 protein expression between AA and NAA TNBC. We hypothesize that the microenvironment of African AA TNBC patients may be different than that of NAAs.

Method: We selected N=43 AA and N=43 NAA TNBC samples from the Yale Pathology archives that were matched by diagnosis date. We measured CD8, CD68 and PD-L1 protein expression in both the tumor and stromal compartments in whole slides from formalin fixed paraffin embedded (FFPE) tissues using multiplexed quantitative immunofluorescence (QIF). The average of each marker expression was calculated in all Fields of View (FOV), and the top 10% brightest FOV of each slide (i.e. hotspot).

Results: The frequency of macrophages, as assessed by the expression of CD68 was significantly higher in AA compared to NAA. This was seen by overall assessment for all FOV (mean NAA=2273au versus mean AA=3627au; p = 0.0052), and in hotspot FOVs (mean NAA=4858au versus mean AA=6371au; p = 0.0411), and also for assessments in tumor and stromal subcompartments. Expression of CD8 positive cytotoxic T cells was significantly lower in AA compared to NAA, but only when measuring hotspots in the tumor compartment (p=0.017).

Conclusion: The significantly higher CD68 and lower CD8 expression in AA compared to NAA TNBC might contribute to a more immune attenuated microenvironment. We are currently further characterizing the macrophage polarity and the cytokine milieu of these samples.

Citation Format: Vesal Yaghoobi, Vasiliki Pelekanou, Tess O'Meara, Andrea Silber, Lajos Pusztai, David Rimm. Vesal Yaghoobi [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 2842.
Abstract 2848: TSPAN8-EPS8L3 signaling play a role in EGF-dependent pancreatic cancer cell invasion and migration
Authors: Li Junjian , Zhu Rongxuan , Ju Gaoda , Ge Weiyu , Jiang Mengyi , Zhou Tianhao , Wang Hongxia
Published: August 2019

The mechanism and function of TSPAN8 on the invasion and metastasis of cancer cells is poorly understood. In present study, we demonstrated that TSPAN8 over-expressed in pancreatic cancer (Paca) tissues (37.98%) comparing with normal tissue (0%). TSPAN8 expression levels were reversely correlated with the overall survival time of Paca patients (p = 0.021). In vitro, over-expressed or knockdown TSPAN8 expression in Paca cells SW1990 or Capan-2 and AsPC-1 modulated invasion, immigration and clone formation ability. Mechanistically, we revealed that SOX9, a downstream transcription factor of EGFR signaling, activated TSPAN8 transcription under EGF condition. Meanwhile, we uncovered a previously uncharacterized mechanism underlying EGF-dependent Paca cells invasion and migration. In brief, TSPAN8 was phosphorylated at Ser129 by AKT, resulting in the binding of TSPAN8 with BCAFL1, TSPAN8 entering nucleus and regulating the EPS8L3 transcription, remodeling of cytoskeleton, and enhancing cell invasion and migration. By single cell seq analysis of Paca cells derived from patients and tissue array assay, we validated that the EGFR and EPS8L3 expression in mRNA and protein level were highly correlated with TSPAN8 expression. In vivo and in vitro study demonstrated that EPS8L3 depletion or overexpression rescued the invasion ability induced by TSPAN8. These findings reveal a molecular basis of EGF-TSPAN8-EPS8L3 signaling and highlight the critical role of TSPAN8 in tumor metastasis.

Citation Format: Junjian Li, Rongxuan Zhu, Gaoda Ju, Weiyu Ge, Mengyi Jiang, Tianhao Zhou, Hongxia Wang. TSPAN8-EPS8L3 signaling play a role in EGF-dependent pancreatic cancer cell invasion and migration [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 2848.
Abstract 2853: Cell motility and invasiveness are promoted by guanylate binding protein 1, GBP-1 in lung adenocarcinoma
Authors: Mimae Takahiro , Yamakita Ichiko , Tsutani Yasuhiro , Miyata Yoshihiro , Ito Akihiko , Okada Morihito
Published: August 2019

Previously, we identified candidate molecules involved in human lung adenocarcinoma progression from non-invasive to invasive tumor by comparing gene expression profiles of each component in the same tumor using laser microdissection and DNA microarray analysis. Among them, guanylate-binding protein 1 (GBP-1) was selected for further analysis because RT-PCR for normal lung and invasive human lung adenocarcinoma tissue samples showed that the relative GBP-1 gene expression levels normalized to GAPDH for invasive lung adenocarcinoma were approximately three-fold higher than those for normal lung samples. In addition, GBP-1 gene and protein expression levels were also higher in mesenchymal-like than in epithelial-like lung cancer cell lines. To determine whether GBP-1 is involved in lung adenocarcinoma cell motility and invasion, we performed migration and wound healing assays using RERF-LC-OK, human lung adenocarcinoma cells transfected with siRNAs. The relative migration of transfected GBP1-siRNA cells was significantly lower than that of control-siRNA cells. The relative wound healing capacities were assessed after scratching. Cells transfected with GBP1-siRNAs were also significantly lower than those of the control-siRNAcells. Moreover, Immunohistochemistry of 80 patients with stage I lung adenocarcinoma demonstrated that non-invasive component cells showed clearly negative GBP-1 expression in all cases. On the other hand, invasive component cells were GBP-1 positive in 10 cases (12.5%) and GBP-1 negative in 70 cases (87.5%). Lymphatic-vascular invasion was positive in 20 patients (25%) and positively correlated with GBP-1 expression (P< 0.05). In conclusion, GBP-1 might enhance lung adenocarcinoma cell motility and invasiveness and controlling of GBP-1 expression has the potential to contribute to the development of novel therapeutic strategies for lung adenocarcinoma.

Citation Format: Takahiro Mimae, Ichiko Yamakita, Yasuhiro Tsutani, Yoshihiro Miyata, Akihiko Ito, Morihito Okada. Cell motility and invasiveness are promoted by guanylate binding protein 1, GBP-1 in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2853.
Abstract 2860: RCP augments oral cancer cell invasion through Zeb1 and Slug expression
Authors: Kim Jin Young , Cho Kyung Hwa , Jeong Bo Young , Song Zi Hae , Park Chang Gyo , Lee Hoi Young
Published: August 2019

Background: Rab coupling protein (RCP), also called family interacting protein 1 (Rab11fip1) is a protein encoded by the RAB11FIP1 gene and has been known to augment cancer tumorigenesis, invasion, and metastasis. Recently, RCP was reported to aggravate cancer cell invasion and metastasis by stabilizing beta1 integrin and inducing epithelial-mesenchymal transition (EMT) in breast and ovarian cancer. Further, a high RCP expression was significantly correlated with shorter overall survival and disease-free survival of HNSCC patients. However, the molecule mechanisms by which RCP increases HNSCC invasion and metastasis are unclear. In the present study, we demonstrate the role of RCP in oral cancer cell invasiveness and its underlying mechanism.

Methods: Oral cancer YD-10B and YD-38 cells were transiently transfected with RCP constructs. siRNAs were used to analyze the role of specific proteins for Slug and Zeb1 expression. Immunoblotting and immunofluorescence were used to analyze protein expression. Matrigel-coated in vitro insert and wound healing assay were used to analyze cancer cell invasion and migration, respectively.

Results: We observed that ectopic expression of RCP induces invasion of oral cancer cells. Conversely, silencing of RCP expression increased E-cadherin expression and reduced oral cancer cell invasion. In addition, RCP upregulated Zeb1 and Slug expression that is important for RCP-induced oral cancer cell EMT and invasion. Furthermore, we identified that silencing of beta1 integrin efficiently attenuates RCP-induced Zeb1 and Slug expression. In addition, we observed that a selective inhibitor of EGFR kinase activity, Gefitinib, significantly reduces RCP-induced Zeb1 and Slug expression and that RasN17 recovers RCP-induced Zeb1 expression. Finally, RCP increased the viability of oral cancer cells against a chemotherapeutic agent cisplatin.

Conclusion: Collectively, we demonstrate that RCP augments oral cancer cell EMT and invasiveness through a beta1 integrin/EGFR/Ras/Slug and Zeb1 signaling axis, providing potential biomarkers for oral cancer patients therapy.

Citation Format: Jin Young Kim, Kyung Hwa Cho, Bo Young Jeong, Zi Hae Song, Chang Gyo Park, Hoi Young Lee. RCP augments oral cancer cell invasion through Zeb1 and Slug expression [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 2860.
Abstract 2866: Surfaceome profiling in osteosarcoma: Identification of the candidate immunotherapeutic target
Authors: Wang Yifei , Zhang Zhongting , Kannan Sankaranarayanan , Zhang Wendong , Roth Michael , Gill Jonathan B. , Xu Zhaohui , Tian Xiangjun , Wang Jing , Gorlick Richard
Published: August 2019

Osteosarcoma (OS) is the most common primary malignant bone tumor in pediatric patients. Current developments in targeted therapeutic strategies show potential clinical implications in cancer treatments. However, immunotherapies such as antibody-drug conjugates (ADCs) or CAR-T cell therapy have not been sufficiently studied in OS because of the lack of tumor-specific target. In this study, we developed a multi-step RNA-seq based pipeline. First, we did RNA sequencing for our OS PDX models and 17 patient-derived OS cell lines. The results are then pooled with the RNQ-seq data from 111 OS patient via the Therapeutically Applicable Research to Generate Effective Treatments project (TARGET). All these tumor data are subsequently compared with normal tissue RNA-sequencing data from NIH Genotype-Tissue Expression (GTEx) database. The significantly differentially expressed genes (log fold change tumor versus normal >1 for each tissue, p < 0.01) are selected. We further filtered this gene list by cell surface protein prediction based on Gene Ontology, the TransMembrane prediction using hidden Markov models (TMHMM), and glycosylphosphatidylinositol (GPI)-anchored protein annotations. Based on the transcriptomic result, ranks of differentially expressed genes were generated. CD276, LCLAT1, and OR8B12 are the surface markers with high ranks. To validate the transcriptomic results, we prepared surface protein extraction of the PDX models and patient-derived OS cell lines. We are profiling these surface proteins by proteomic mass spectrometry. Further validation for the expression level and location of these markers are underway. Our current finding is not previously reported in OS. The results warrant further development of ADC and car-T cell therapy.

Note: This abstract was not presented at the meeting.

Citation Format: Yifei Wang, Zhongting Zhang, Sankaranarayanan Kannan, Wendong Zhang, Michael Roth, Jonathan B. Gill, Zhaohui Xu, Xiangjun Tian, Jing Wang, Richard Gorlick. Surfaceome profiling in osteosarcoma: Identification of the candidate immunotherapeutic target [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 2866.
Abstract 2869: Zika virus is a potent oncolytic agent against aggressive human ependymoma
Authors: Kaid Carolini , Semedo-Kuriki Patrícia , Goulart Ernesto , Caires-Júnior Luiz , Astray Renato , Okamoto Oswaldo Keith , Zatz Mayana
Published: August 2019

Ependymoma is a metastatic CNS tumor responsible for 10% of all pediatric brain tumors. Displaying a poor survival rate of only 32.5%, the conventional ependymoma treatment is surgery and radiation therapy associated to significant morbidity. Previous studies found significantly increased expression of NESTIN, a neural progenitor cell (NPC) marker, in aggressive ependymoma. Zika virus (ZIKV) has recently emerged as an oncolytic therapy against adult and pediatric CNS tumors due to its ability to infect NPCs and neural stem-like tumor cells. We showed for the first time that Brazilian Zika virus strain efficiently destroyed pediatric CNS tumor cells in vitro and in vivo, resulting in significantly mice longer survival and fewer metastasis. Once tumor cells overexpressing NPCs markers are more susceptible to ZIKV infection, here we evaluated the oncolytic properties of Brazilian ZIKV against human ependymoma. A patient-derived ependymoma cell line (USP21-EPE) has been established and found to abnormally express the pluripotency markers CD133, SOX2 and NESTIN. USP21-EPE monolayer culture showed massive cell death three days after ZIKV infection in four tested MOIs: 0.01, 0.1, 1 and 2. At MOI 0,1 50% of cell population decreased viability while at higher MOIs was observed 100% of cell death 72 hours post ZIKV infection. Moreover, ZIKV significantly disrupted CNS ependymoma tumorspheres reinforcing its oncolytic effect against the ependymoma tumor cells. Considering the poor effectiveness and severe side effects of available treatments for ependymoma and that most ZIKV infections are associated to very mild or no symptoms, our findings open new avenues for novel therapies against this aggressive pediatric tumor.

Citation Format: Carolini Kaid, Patrícia Semedo-Kuriki, Ernesto Goulart, Luiz Caires-Júnior, Renato Astray, Oswaldo Keith Okamoto, Mayana Zatz. Zika virus is a potent oncolytic agent against aggressive human ependymoma [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 2869.
Abstract 2871: Global reduction of H3K4me3 improves chemotherapeutic efficacy for pediatric ependymomas
Authors: Xi Guifa , Wadhwani Nitin , Hashizume Rintaro , Mania-Farnell Barbara , Soares Marcelo Bento , James Charles D. , Tomita Tadanori
Published: August 2019

Background Ependymomas (EPNs) are the third most common brain tumor in children. These tumors are resistant to available chemotherapeutic treatments, therefore new effective targeted therapeutics must be identified. Increasing evidence shows epigenetic alterations including histone posttranslational modifications (PTMs), are associated with malignancy, chemotherapeutic resistance and prognosis for pediatric EPNs. In this study we examined histone PTMs in EPNs and identified potential targets to improve chemotherapeutic efficacy.

Methods Global histone H3 lysine 4 trimethylation (H3K4me3) levels were detected in pediatric EPN tumor samples with immunohistochemistry and immunoblots. Candidate genes conferring therapeutic resistance were profiled in pediatric EPN tumor samples with micro-array. Promoter H3K4me3 occupancy was examined for two candidate genes, CCND1 and ERBB2, with chromatin-immunoprecipitation coupled with real-time PCR (ChIP-PCR). These methods and MTS assay were used to verify a relationship between H3K4me3 levels and CCND1 and ERBB2, and to investigate cell viability in response to chemotherapeutic drugs in primary cultured pediatric EPN cells.

Results H3K4me3 levels positively correlate with WHO grade malignancy in pediatric EPNs and are associated with progression free survival in patients with posterior fossa group A EPNs (PF-EPN-A). Reduction of H3K4me3 by silencing its methyltransferase SETD1A, in primary cultured EPN cells, increased cell response to chemotherapy.

Conclusions Our results support the development of a novel treatment that targets H3K4me3 to increase chemotherapeutic efficacy in pediatric PF-EPN-A tumors.

Key words: promoter, H3K4me3, human SETD1A, chemotherapy, pediatric ependymoma

Citation Format: Guifa Xi, Nitin Wadhwani, Rintaro Hashizume, Barbara Mania-Farnell, Marcelo Bento Soares, Charles D. James, Tadanori Tomita. Global reduction of H3K4me3 improves chemotherapeutic efficacy for pediatric ependymomas [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 2871.
Abstract 2874: De novo acquisition of RD3 loss with intensive multi-modal therapy harmonize neuroblastoma relapse
Authors: Somasundaram Dinesh Babu , Subramanian Karthikeyan , Aravindan Sheeja , Yu Zhongxin , Natarajan Mohan , Herman Terence S. , Aravindan Natarajan
Published: August 2019

The majority of high-risk neuroblastoma that initially responds to therapy will ultimately relapse. Acquired genetic/molecular rearrangement contributes to tumor relapse and currently, no salvage treatment regimens are known to be curative. Our studies identified the loss of retinal-degeneration-protein-3 (RD3) in progressive disease (PD) and its association with poor clinical outcomes. Herein we investigated the acquisition of RD3-loss after current standard of care (SOC, intensive multimodal therapy) in neuroblastoma. Acquired transcriptional (RNA in situ hybridization, QPCR) and translational (IHC on a custom archived cell micro array, immunoblotting) loss of RD3 with SOC was investigated with bed-to-bench approach utilizing a panel of 15 stage-4 patient derived cell-lines with known therapy status, diagnosis [DX] vs. PD after SOC). Further, RD3-loss in progressive disease, its direct role in the pathogenesis disease progression (cell migration, invasion, tumorosphere formation and metastatic potential) was investigated using mouse model of progressive neuroblastoma and in experimental in vitro and ex vivo settings coupled with gene manipulation strategies. In addition, RD3 status in response to therapy were also affirmed by in silico analysis of independent studies on bed-to-bench and experimental models. Results demonstrated RD3-loss (transcriptional/translational) acquisition with SOC in human PD. Experimental in vitro, in vivo, ex vivo studies affirmed RD3 loss in PD and, further forced RD3-rearrangements in these models defined its functional role in PD pathogenesis. Data-mining experimental studies with salvage therapeutic agents affirmed the acquisition of RD3-loss in resistant cells and in residual tumors. For the first time, these results demonstrate the de novo acquisition of RD3 loss in PD after intensive multi-modal therapy. Defining the criticality of RD3-loss in PD and the defined role of RD3 loss in PD pathogenesis, benefit of targeted reinforcement could lead to improved salvage therapy for high-risk neuroblastoma.

Citation Format: Dinesh Babu Somasundaram, Karthikeyan Subramanian, Sheeja Aravindan, Zhongxin Yu, Mohan Natarajan, Terence S. Herman, Natarajan Aravindan. De novo acquisition of RD3 loss with intensive multi-modal therapy harmonize neuroblastoma relapse [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 2874.
Abstract 2876: p21-activated kinase 1 is a novel therapeutic target for metastatic osteosarcoma harboring p27 mislocalization
Authors: Chen Xiang , Cates Justin , Du Yuchen , Sung Yun Jung , Lo Xiao-nan , Hicks John , Man Tsz-Kwong
Published: August 2019

Lung metastasis is the leading cause of death in osteosarcoma (OS), the most common bone cancer in children and young adults. Due to a lack of biomarkers and therapeutic targets, significant progress has not been made in the treatment of metastatic OS over the past three decades. The survival of metastatic patients remains dismal. We have previously reported that the tumor suppressor protein p27 (KIP1, CDKN1B) is frequently mislocalized in the cytoplasm of OS, which promotes the motility and invasiveness of the tumor cells and the development of pulmonary metastases in mice. In this study, we showed that a higher proportion score of cytoplasmic p27 significantly correlated with metastases developed within 3- or 5-year of follow-up, and poor disease-specific and event-free survival in a large OS cohort (n=136). Using immunoprecipitation followed by mass spectrometry, we demonstrated that cytoplasmic p27 interacted with p21-activated kinase 1 (PAK1), where the two proteins co-localized in the cytoplasm of OS cells. PAK1 is a serine/threonine kinase that has been implicated in various cancers; however, its role in OS is still elusive. We showed that the phosphorylations at serine-144 and threonine-423 residues of PAK1 were higher in the p27-mislocalized cells relative to the control cells. The PAK1 phosphorylations increased actin stress fiber formation in the tumor cells. Gene silencing or therapeutic inhibition of PAK1 reduced the motility of p27-mislocalized cells in vitro. Similar results were observed in other cancer cell lines known to have the p27 mislocalization, suggesting PAK1 is a common and potential downstream effector of p27-mediated metastasis. An experimental metastasis mouse model further showed that a PAK1-silenced mutant had a significant impairment in the development of pulmonary metastasis. In summary, our study indicates that cytoplasmic p27 is a novel prognostic biomarker in OS. In the tumor cell, the mislocalization activates PAK1-mediated cell motility and metastasis. Targeting PAK1 in metastatic OS harboring the p27 mislocalization may be exploited as a potential therapeutic intervention to improve the poor outcome of the affected children.

Citation Format: Xiang Chen, Justin Cates, Yuchen Du, Yun Jung Sung, Xiao-nan Lo, John Hicks, Tsz-Kwong Man. p21-activated kinase 1 is a novel therapeutic target for metastatic osteosarcoma harboring p27 mislocalization [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 2876.
Abstract 2878: CRISPR-Cas9 screens identify the nuclear export factor NXT1 as a novel therapeutic target in MYCN-amplified neuroblastoma
Authors: Malone Clare F. , Dharia Neekesh V. , Kugener Guillaume , Paolella Brenton , Rothberg Michael , Abdusamad Mai , Gonzalez Alfredo , Dumont Nancy , Younger Scott , Root David , Vazquez Francisca , Stegmaier Kimberly
Published: August 2019

Pediatric cancers, such as neuroblastoma, have a relative lack of oncogenic mutations and the known drivers of these cancers are largely transcription factors, a target class notoriously difficult to “drug.” In order to identify novel therapeutic targets for high-risk, MYCN-amplified neuroblastoma, we employed functional genomic screening using a CRISPR-Cas9 approach. We generated genome-scale CRISPR dependency data to identify a candidate gene list of 197 putative genetic dependencies in neuroblastoma. We next created a focused sgRNA library targeting these genes and performed time-course dropout screens using CRISPR and CRISPRi and Annexin-V-based positive-selection cell death screens in four MYCN-amplified neuroblastoma cell lines. We also screened this library in vivo in two xenograft models of MYCN-amplified neuroblastoma. At the intersection of these screens, we identified the nuclear export factor NXT1 as a top candidate for therapeutic development in neuroblastoma. NXT1 scores as a strong dependency using both CRISPR and CRISPRi, is strongly enriched in our Annexin-V based positive selection cell death screen and scores in our in vivo screen. We have validated that NXT1 is indeed a genetic dependency in neuroblastoma in low-throughput and that NXT1 loss induces apoptosis in these cell lines. We were next interested in identifying biomarkers of dependency on NXT1, and by integrating RNAseq data with CRISPR dependency data, we identified low expression of NXT2, a paralog of NXT1, as the top predictive feature of NXT1 dependency. Importantly, while NXT1 itself is not currently druggable, inhibitors against another nuclear export protein, CRM1, are currently in Phase II clinical trials, demonstrating this class of proteins has the potential to be effectively drugged. Together, we show that CRISPR-Cas9 functional screens can be used to identify new therapeutic targets, particularly in diseases that lack “druggable” oncogenic drivers, such as many pediatric cancers.

Citation Format: Clare F. Malone, Neekesh V. Dharia, Guillaume Kugener, Brenton Paolella, Michael Rothberg, Mai Abdusamad, Alfredo Gonzalez, Nancy Dumont, Scott Younger, David Root, Francisca Vazquez, Kimberly Stegmaier. CRISPR-Cas9 screens identify the nuclear export factor NXT1 as a novel therapeutic target in MYCN-amplified neuroblastoma [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 2878.
Abstract 2979: Boosting replication and penetration of telomerase-specific replicative virus by paclitaxel induces synthetic lethality in peritoneal metastasis of gastric cancer
Authors: Ogawa Toshihiro , Kikuchi Satoru , Ishikawa Wataru , Tazawa Hiroshi , Tabuchi Motoyasu , Kuroda Shinji , Noma Kazuhiro , Nishizaki Masahiko , Kagawa Shunsuke , Urata Yasuo , Fujiwara Toshiyoshi
Published: August 2019

Background: Peritoneal metastasis is the most frequent form of distant metastasis and recurrence in gastric cancer and its prognosis is extremely poor. A promising result in phase III trial using intraperitoneal (i.p.) paclitaxel (PTX) for the peritoneal metastasis of gastric cancer has been reported in recent year; however, it is not sufficiently effective to eradicate disseminated gastric cancer. In this study, we investigated whether novel i.p. oncolytic virotherapy could be synthetically lethal with PTX for the peritoneal metastasis of gastric cancer.

Methods: OBP-401 (TelomeScan) is a green fluorescence protein (GFP)-expressing attenuated adenovirus with oncolytic potency that is driven by telomerase promoter. We assessed its synergistic effect with PTX using human gastric cancer cell lines (GCIY and KATO III) and xenograft peritoneal metastasis model. The mechanism underlying the synergistic antitumor effect was evaluated by in vitro viral replication, induction of mitotic catastrophe, and in vivo viral penetration into disseminated nodules.

Results: OBP-401 synergistically suppressed the viability of human gastric cancer cells in combination with PTX. PTX was synthetically lethal with OBP-401 by enhancing viral replication ability in cancer cells and the combination therapy increased mitotic catastrophe induction, resulted in accelerating autophagy and apoptosis. Peritoneally disseminated nodules were selectively visualized as GFP-positive spots by the i.p. administration of OBP-401 in the orthotopic human gastric cancer peritoneal dissemination model. And, PTX enhanced the penetration of OBP-401 deeply into the disseminated nodules. Moreover, non-invasive in vivo imaging system (IVIS) demonstrated that the combination therapy of i.p. OBP-401 administration with PTX significantly inhibited tumor growth of peritoneal metastasis and the amount of malignant ascites.

Conclusions: Intraperitoneal virotherapy with PTX may be a promising treatment strategy for the peritoneal metastasis of gastric cancer. Clinical trials to obtain the proof of concept are warranted in peritoneally disseminated gastric cancer patients.

Citation Format: Toshihiro Ogawa, Satoru Kikuchi, Wataru Ishikawa, Hiroshi Tazawa, Motoyasu Tabuchi, Shinji Kuroda, Kazuhiro Noma, Masahiko Nishizaki, Shunsuke Kagawa, Yasuo Urata, Toshiyoshi Fujiwara. Boosting replication and penetration of telomerase-specific replicative virus by paclitaxel induces synthetic lethality in peritoneal metastasis of gastric 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 2979.
Abstract 3: Discovery and optimization of small molecule Bax activators for cancer therapy
Authors: Liu Gang , Kim Hyejin , Ding Chunyong , Yu Zhuo , Wang Hong , Chen Haiying , Shen Qiang , Zhou Jia
Published: August 2019

Bax, an indispensable executioner protein of B-cell lymphoma 2 (Bcl-2) family, plays a pivotal role in controlling the mitochondrial dysfunction and apoptosis in normal and cancer cells. High throughput screening to combat the nicotine-induced Bax phosphorylation at serine 184 (S184) to activate the proapoptotic function of Bax has revealed compound SMBA1 as a promising Bax direct activator as we previously reported. Initial medicinal chemistry campaign by choosing SMBA1 as a lead compound for breast cancer treatment resulted in the identification of compound CYD-2-11 with low micromolar IC50 values against MDA-MB-231 and MCF-7 as well as in vivo inhibitory effects against MDA-MB-231 xenograft tumor growth. Herein, we report the comprehensive structural optimization based on CYD-2-11 as the lead by introducing various alkylamino side chains to have a deeper access to S184 pocket and replacing carbon atoms with nitrogen as well as reducing the nitro group of 9H-fluorene scaffold to construct diverse derivatives for enhanced drug properties. CYD-4-61 and GL0385 have been discovered as the most potent anti-breast cancer agents with submicromolar IC50 values of 0.34 and 0.33 µM against MDA-MB-231 as well as 0.65 and 0.26 µM against MCF-7, respectively. More importantly, these drug candidates exhibited significant in vivo efficacy suppressing xenograft tumor growth by activating Bax to induce cancer cell apoptosis with the potential to overcome resistance against Adriamycin (ADR) in breast cancer. This work was supported by Breast Cancer Research Program (BCRP) Breakthrough Award BC160038 (to J.Z.) and BC160038P1 (to Q.S.) from the Department of Defense (DoD).

Citation Format: Gang Liu, Hyejin Kim, Chunyong Ding, Zhuo Yu, Hong Wang, Haiying Chen, Qiang Shen, Jia Zhou. Discovery and optimization of small molecule Bax activators 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 3.
Abstract 300: A new combination therapy to treat metastatic breast cancer
Authors: Sharif-Askari Bahram , Aloyz Raquel , Panasci Lawrence
Published: August 2019

Poly (ADP-ribose) polymerase (PARP) resulting poly (ADP-ribosyl)ation is an early player in modification of proteins detected at single strand breaks and double strand breaks contributing to DNA repair. PARP1, 2 and 3 are involved in this process. However, PARP3 is unique in that, it is also required for efficient mitotic progression, specifically in stabilization of the mitotic spindle. The vinca alkaloids such as vinorelbine, arrest cells in metaphase due to inhibition of the assembly and dynamics of microtubules. Nontoxic concentrations of 2 PARP3 inhibitors, ME-0328 and olaparib, sensitized various her-2 negative and triple negative breast cancer cell lines greater than or equal to 10 fold to vinorelbine associated with potentiation of vinorelbine’s interaction with tubulin and also vinorelbine-induced PARP3 inhibition, mitotic arrest, and apoptosis without evidence of a DNA repair mechanism involved in this sensitization (Sharif-Askari B et al. Breast Cancer Res Treat. 2018 Nov;172(1):23-32).

Eribulin is a Halichondrin B analogue, an antitubulin drug, with a unique mechanism of action; it inhibits microtubule dynamics via a novel mechanism of action which seems to involve binding to a unique site on tubulin resulting in the suppression of microtubule polymerization without affecting depolymerization along with sequestration of tubulin into nonfunctional aggregate. After impressive results in phase III trials (including one of the rare trials to show prolongation of survival in comparison to standard of care in heavily pretreated metastatic breast cancer patients) the U.S. Food and Drug Administration approved eribulin for treatment of patients with metastatic breast cancer. PARP3 inhibitors again sensitized at least 5 fold her-2 negative and triple negative breast cancer cell lines to eribulin. We will do similar studies with eribulin as we have done with vinorelbine including in-vivo studies. These studies could define a new therapy in the treatment of metastatic triple negative and ER+/her-2 negative breast cancer. The inhibition of PARP3 may increase the sensitivity of tumor cells to mitotic spindle poison chemotherapies. Our results should stimulate development of specific PARP3 inhibitors for clinical use.

Citation Format: Bahram Sharif-Askari, Raquel Aloyz, Lawrence Panasci. A new combination therapy to treat metastatic 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 300.
Abstract 3006: A novel strategy of targeting opioidergic receptors to potentially reduce human breast cancer cell proliferation, colony formation, invasion, and migration
Authors: Murugan Sengottuvelan , Patel Vanisha , Sarkar Dipak K.
Published: August 2019

Breast cancer is the second most prevalent cancer after lung cancer among American women. The efficacy of current antitumor therapies is still quite limited. Endogenous opioid peptides, such as endorphin, which is involved in body stress controlling mechanisms, have been shown to enhance immune function and prevent cancer growth and progression in various animal models of cancers. Therefore, targeting the endogenous opioid peptides receptors in the regulation of cancer chemoprevention may offer new promise for the design of therapeutic strategies. Our previous studies have shown differential actions of mu-opioid receptor (MOR) and delta opioid receptor (DOR) on various cell types. In this study we tested the effects of a MOR antagonist naltrexone (NTX) and a DOR agonist (D-Ala2-,N-Me-Phe4,Gly-ol Enkephalin; DPDPE) on proliferation and invasion of human breast cancer cells of different molecular subtype such as T47D (Luminal A), MDA-MB-231 (triple negative, Claudin-Low) and MDA-MB- 468 (triple negative, basal A) in cultures. We used cytotoxicity, clonogenic, cell migration and cell invasion assays to identify effects of these drugs on cancer cells. We found that both NTX and DPDPE have significant cytotoxic effects on these breast cancer cells. Our work from clonogenic, cell migration and cell invasion assays showed that both NTX and DPDPE inhibit the formation of colony, migration and invasion of cancer cells in a concentration-dependent manner. These data suggest that naltrexone and DPDPE have anticancer efficacy in subtypes of human breast cancer cells. The anticancer effects varied depending on the concentration of the drugs and the subtype of breast cancer cells. (Supported by NIH grant R01 CA20863201).

Citation Format: Sengottuvelan Murugan, Vanisha Patel, Dipak K. Sarkar. A novel strategy of targeting opioidergic receptors to potentially reduce human breast cancer cell proliferation, colony formation, invasion, and migration [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 3006.