bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4910: Neurocognitive effects of central nervous system directed chemotherapy in Non Hodgkin Lymphoma diseased children
Authors: Elmoneim Abeer Abd , Eladawy Ahlam , Elkasem Mona Abu , Hadhood Soumaya
Published: August 2019

Background and Purpose. Because the blood-brain barrier serves as a pharmacological barrier, malignant cells can remain in the central nervous system (CNS) despite systemic chemotherapy. Consequently, CNS-directed treatment is an essential part of therapy for Non-Hodgkin lymphoma (NHL). We aimed in this analysis to determine the neurocognitive effects of CNS-directed chemotherapy in children with NHL.

Methods.

In a cross-sectional study 20 NHL diseased children and 10 healthy controls were tested for neurocognitive functions. The selection criteria were: 3 years or more after the end of NHL chemotherapy only regimen, no anaemia, no CNS or systemic diseases. Both patients and healthy controls were examined for global intelligence quotient (IQ), verbal IQ, performance IQ, attention measures, auditory tests, long and short memory, math skills and academic achievement.

Results.

No decline in global IQ in NHL children compared to healthy controls but there was a significant decline of attention, verbal comprehension and performance IQ. Among 20 NHL diseased children 18 had poor recent and immediate memories while remote memory was normal in 16 children. Young age at diagnosis and treatment intensity were the commonest risk factors.

Conclusion.

In absence of cranial irradiation, childhood survivors of NHL experience long-term neurocognitive deficits after chemotherapy treatment. Global IQ is not a sufficiently sensitive measure to detect specific CNS deficits in NHL diseased children. The deficits are mainly present in basic neurophysiologic processes of attention and executive functioning.

Note: This abstract was not presented at the meeting.

Citation Format: Abeer Abd Elmoneim, Ahlam Eladawy, Mona Abu Elkasem, Soumaya Hadhood. Neurocognitive effects of central nervous system directed chemotherapy in Non Hodgkin Lymphoma diseased children [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 4910.
Abstract 4915: Optimal frequency of follow up scans on systemictherapy for advanced malignancies
Authors: Stewart David J. , Macdonald Blair , Katwyk Sasha Van , Awan Arif , Thavorn Kednapa
Published: August 2019

Background: Frequency of follow-up scans is specified in trials but there is little evidence to guide optimal frequency in standard therapy. Progression-free survival (PFS) generally follows first order kinetics and PFS half-lives (calculated by nonlinear regression analysis of more than 300 published PFS curves) correlated strongly with PFS medians (Stewart, AACR 2018).

Method: We used the Excel formula EXP(-tn*0.693/t1/2) to calculate proportion of residual patients remaining progression-free at different time intervals, where tn is the potential time of follow-up scans (eg, every 3 weeks, 6 weeks, etc), * indicates multiplication, 0.693 is the natural logarithm of 2, and t1/2 is the PFS half-life in weeks.

Results: Proportion of remaining patients expected to still remain progression-free at each subsequent scan varied with time interval between scans and with PFS half-life for the individual regimen. For example, with a PFS half-life of 4 months (17.3 weeks) and scans repeated every 6 weeks, 21% of the patients would have progressed by the first scan, 21% of the remaining patients would have progressed by the second scan at 12 weeks, etc. With PFS half-lives of 2, 4, 6, 12 and 20 months (for example), the proportion of remaining patients progressing by the time of each subsequent follow up scan if the scans were repeated every 3 weeks would be 21%, 11%, 8%, 4% and 2%, respectively, while with scans repeated every 6 weeks the proportion progressing would be 38%, 21%, 15%, 8% and 5%, by 12 weeks it would be 62%, 38%, 27%, 15% and 9%, and by 15 weeks it would be 70%, 45%, 33%, 18% and 11%, respectively.

Conclusions: For therapies with short PFS half-lives, scanning every 6 weeks may not be often enough, while with long PFS half-lives, less frequent scans may be warranted. We plan analyses to estimate economic implications of varying scan frequency based on PFS half-life, drug and toxicity costs, opportunity costs (where other effective therapies might be offered if progression is detected), etc. We will also assess how this approach might be adapted for PFS curves that follow exponential 2-phase decay due to presence of distinct good vs poor prognosis subgroups, and we are in the process of using published PFS curves to assess the PFS half-lives for multiple different cancer therapies.

Citation Format: David J. Stewart, Blair Macdonald, Sasha Van Katwyk, Arif Awan, Kednapa Thavorn. Optimal frequency of follow up scans on systemictherapy for advanced malignancies [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 4915.
Abstract 5124: A machine learning based approach to identify biomarkers of environmental toxicant exposures relevant to liver cancer disparities in rural Illinois
Authors: Smith Brandi , Madak-Erdogan Zeynep
Published: August 2019

Although there have been nationwide reductions in recent cancer death, liver cancer mortality remains to be a problem in rural Illinois. Researchers anticipate that exposure to environmental toxicants in rural Illinois, may be possible causes of increased liver cancer mortality in females. The purpose of our study is to identify potential biomarkers, which are related to early liver toxicity through machine learning. We accessed gene expression data from the open source Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System (TG-GATES) database from rats that had been exposed to over 170 toxicants relevant to liver cancer. We performed a differential gene expression analysis to identify significant features or genes, which are related to hepatotoxicity endpoints, specifically necrosis. Then we built our predictive model using supervised machine learning (ML) through feature selection and classification models. We performed differential gene expression analysis and feature selection to decrease the dimensionality of the gene set. We then used classification modeling to classify genes related to necrosis. Lastly, we tested our predictive model, on an independent data set. Our model predicted specific genes that were highly related to liver toxicant exposure.

Note: This abstract was not presented at the meeting.

Citation Format: Brandi Smith, Zeynep Madak-Erdogan. A machine learning based approach to identify biomarkers of environmental toxicant exposures relevant to liver cancer disparities in rural Illinois [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 5124.
Abstract 5125: A novel method for isolating high-quality UHMW DNA from 10 mg of freshly frozen or liquid-preserved animal and human tissue including solid tumors
Authors: Zhang Yang , Broach James
Published: August 2019

High-quality UHMW DNA is key to success in optical mapping of long genomic DNA using Bionano Genomics Saphyr® system for de novo genome assembly and structural variation detection. Ideally, ~100 mg of tissue is frozen immediately upon collection using liquid nitrogen or dry ice, and stored at -80°C before DNA isolation. Practically, tissue amounts may be limited and collection often occurs at sites where low temperature preservation for storage and shipping may be unavailable.

Here we present a novel method to isolate UHMW DNA from 10 mg of either freshly frozen or room temperature preserved animal and human tissue for subsequent enzymatic labeling using direct labeling and staining (DLS) to generate Bionano maps. With a TissueRuptor, 10 mg tissue is homogenized followed by a three-step purification process before embedding the purified nuclei in agarose gel plugs. This process takes less than 3 hours for a batch of 6 samples. Using this method, we have successfully isolated high quality UHMW DNA from various tissue types of rat and human including solid tumors.

The method is very attractive compared to other protocols because: 1) tissue preservation, shipping and handling can be done at room temperature, 2) uses very small amount of tissue - possible application for rare samples and human tissue biopsy tests, and 3) the length and quality of the resulting single molecules generate high quality optical maps for genome finishing and SV calling.

Citation Format: Yang Zhang, James Broach. A novel method for isolating high-quality UHMW DNA from 10 mg of freshly frozen or liquid-preserved animal and human tissue including solid tumors [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 5125.
Abstract 5173: Development of a novel tunable synthetic 3D hydrogel platform for the study of tumor and stromal cell interactions
Authors: Wang Hsu-Kun , Chu Vi , Asbrock Nick
Published: August 2019

Numerous cancer cell models exist used to investigate disease mechanisms and to screen potential cancer therapeutics. Roughly 90% of promising preclinical drugs fail to result in efficacious human treatments. Traditional two-dimensional (2D) tissue culture models lack realistic complexity, while animal models are expensive, time consuming, and too frequently fail to reflect human tumor biology. Recently, three-dimensional (3D) cell culture models are a new method to generate new drug candidates before moving to expensive and time-consuming animal models. We have developed a biochemically defined hydrogel platform formed by mixing various polymers with chemical crosslinkers with enhanced functionality. The hydrogel system employs one of two types of backbone polymers: a synthetic non-degradable polyvinyl alcohol (PVA) or an enzymatically-degradable dextran. Both polymers are functionalized either with fast or slow thiol-reactive groups. Crosslinkers consist of either PEG non-cell-degradable or a CD cell-degradable crosslinkers containing peptide sequences that create cleavage sites for matrix metalloproteases (MMPs) that allow cell migration. The technology provides mechanical and biochemical cues to investigate both morphological and physiological properties of cells in a 3D environment. The hydrogel allows precise control over hydrogel stiffness, gelation speed, cell migration and allows cell recovery for downstream applications. Here, we demonstrate the utility of this hydrogel platform to grow epithelial, fibroblast and tumor cells in 3D cell cultures with high cell viabilities and functionalities. Furthermore, we have constructed a 3D tumor/stromal cell co-culture using the hydrogel to model the dynamic tumor cell microenvironment. This technology will allow the creation of more accurate 3D cancer cell models for basic research and drug discovery applications.

Note: This abstract was not presented at the meeting.

Citation Format: Hsu-Kun Wang, Vi Chu, Nick Asbrock. Development of a novel tunable synthetic 3D hydrogel platform for the study of tumor and stromal cell interactions [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 5173.
Abstract 5175: Expression profile and role of LOXL3 in astrocytomas
Authors: Laurentinho Talita de Souza , Soares Roseli da Silva , Marie Suely Kazue , Oba-Shinjo Sueli Mieko
Published: August 2019

Astrocytomas are the most common primary central nervous system tumors in adults. The WHO classification based on histologial characteristics according consider four malignant grades, glioblastoma (GBM) being the most malignant (grade IV). Molecular classification of GBM is based on the integration of somatic mutations, DNA methylation and specific transcript/protein expression and consider four subtypes: proneural, classic, mesenchymal and neural. Lysyl oxidase family is composed of five members (LOX, LOXL1, LOXL2, LOXL3 and LOXL4) which are enzymes responsible for catalyzing lysine-derived cross-links of collagen and elastin. The aim of the present work was to analyze the gene expression of LOXL3 in astrocytomas of different grades and different GBM molecular subtypes, the impact of LOXL3 expression level on overall survival in GBM cases of our cohort. These data were also analyzed in the TCGA database to validate our findings. LOXL3expression increased with astrocytoma malignant grades, and proved to be a prognostic factor, as GBM patients with lower LOXL3 expression presented longer survival time than those with higher expression (p<0.041). Analogous data was obtained in the TCGA database, when considering temozolamide-treated GBM patients (p=0.049). Additionally, LOXL3 expression was higher in mesenchymal GBM subtype than in proneural and classic GBM subtypes in TCGA cohort (p<0.001). To further understand LOXL3 functional role in gliomagenesis, LOXL3 was silenced with two distinct siRNA sequences in U87MG, a mesenchymal subtype of GBM cell line. LOXL3 down regulation was confirmed at gene and protein expression levels. Functional assays with silenced LOXL3 demonstrated a decrease in the cell proliferation (p<0.05), and an increase in the apoptosis associated to temozolamide treatment (p<0.05). Immunofluorescence analysis in U87MG cells showed LOXL3 colocalized with mitochondria. Interestingly, such colocalization decreased when LOXL3 was downregulated, associated to a clear morphological alteration in mitochondria shape. A rough increase in mitochondria volume was observed, suggesting fused mitochondria. In addition, such condition was associated to a 23% decrease in mitochondrial DNA copy number. Altogether, our findings suggest that LOXL3 is upregulated in GBM, with impact in prognosis and might play a role in mitochondria dynamics and/or mitophagy, modulating apoptosis. Transcriptome analysis, currently in progress, will further clarify the associated players in this signaling pathway.

Citation Format: Talita de Souza Laurentinho, Roseli da Silva Soares, Suely Kazue Marie, Sueli Mieko Oba-Shinjo. Expression profile and role of LOXL3 in astrocytomas [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 5175.
Abstract 5184: N-CoR2 epigenetically regulates adhesion-dependent branching morphogenesis and its loss correlates with malignant progression of the mammary gland
Authors: Huang Shenq-Shyang , Weaver Valerie M. , Tsai Kelvin K.C.
Published: August 2019

Branching morphogenesis depends upon extracellular matrix remodeling and dynamic cell-cell and cell-extracellular matrix interactions, although how this developmental program is coordinated remains unclear. Expression profiling of mammary tissue at different stages of branching morphogenesis combined with histological and functional studies identified the histone deacetylase (HDAC) regulator, nuclear receptor co-repressor 2 (N-CoR2), as a key orchestrator of adhesion-dependent branching morphogenesis in the mammary gland. Molecular studies revealed that N-CoR2 actively represses fibronectin (FN) and thrombospondin-1 (THBS-1) promoter accessibility in an HDAC-dependent manner. Consistently, histological analysis revealed that N-CoR2 is abundantly expressed in the luminal epithelial cells localized along the outer rim of invading terminal end buds (TEB) and that its expression is lowest at sites of TEB branching bifurcation. In addition, reducing N-CoR2 expression drove the scattering of human mammary acini in vitro and promoted promiscuous branching of humanized mouse mammary tissue in vivo, suggesting that downregulation of N-CoR2 is required for branching morphogenesis in mammary gland. Furthermore, downregulation of N-CoR2 in breast cancer cells increased the expression of both FN and THBS-1 and reduced the expression of E-cadherin and enhanced the migratory capability of breast cancer cells. Intriguingly, immunohistochemical and clinical correlative analysis indicated that loss of N-CoR2 correlates with perturbed cell-cell and enhanced cell-ECM adhesion and acquisition of an invasive phenotype and local lymph node metastasis in a subset of breast carcinomas. These findings suggest N-CoR2 may epigenetically regulate adhesion-dependent branching morphogenesis in the mammary gland and that its loss could promote malignant progression of some breast cancers.

Citation Format: Shenq-Shyang Huang, Valerie M. Weaver, Kelvin K.C. Tsai. N-CoR2 epigenetically regulates adhesion-dependent branching morphogenesis and its loss correlates with malignant progression of the mammary gland [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 5184.
Abstract 5193: Conditional knockout of histone chaperone FACT in mice: Consequences for normal tissues
Authors: Ito Masaki A. , Sandlesh Poorva , Goswami Imon , Prendergast Laura A. , Gurova Katerina
Published: August 2019

Histone chaperone FAcilitates Chromatin Transcription (FACT) is, a complex of SSRP1 and SPT16, involved in chromatin remodeling during transcription, replication, and DNA repair. FACT is selectively expressed during early stages of development and later exclusively present in the stem cell niches in adult normal tissues. It is expressed in undifferentiated aggressive tumors with poor prognosis.¹ FACT inhibition is not tolerated by most tumor, unlike normal cells. This vulnerability of tumor cells upon FACT inactivation suggests evaluating FACT inhibition as an antitumor therapeutic approach.¹ Since FACT knockout (KO) is embryonically lethal (E3.5),² study of FACT’s function in different non-tumor cells of mammalian organisms was not possible and was mostly studied in single cell organisms and in tumor cell lines. Therefore, to assess the safety of FACT inhibition as an anticancer approach, by understanding its role in normal physiology, we generated a conditional KO of Ssrp1 mouse model using CreERT2-LoxP system.³ We harvested stem cells of different lineages to better understand the role of FACT in normal mouse physiology. Using the hematopoietic stem cells (HSCs) from the bone marrow (BM), we performed BM colony formation assay. We found that FACT inactivation significantly decreased the number of colonies of HSCs. Furthermore, inhibiting FACT significantly increased caspase 3 expression in mouse adipose stem cells (ASCs), resulting in apoptosis and decreased survival. These results prompted us to comprehensively evaluate roles of FACT in stem cell development and survival. To this end, fibroblasts in a conditional knockout mouse were reprogrammed by retroviral vector encoding four pluripotency transcription factors (Oct3/4, Klf4, Sox2 and c-Myc). We have found successful generation of induced pluripotent stem (iPS) cells 14 days after reprogramming. In conclusion, FACT associates with development and/or survival of mouse HSCs and ASCs. Further evaluation using iPS cells is expected to delineate roles of FACT in various stages of stem cell development and thus normal physiology. 1. Garcia H, et al. Cell Rep, 2013 4(1) 2. Cao S, et al. Mol Cel Bio. 2003 23(15) 3. Sandlesh P et al. PLOS ONE. 2018 13(6)

Citation Format: Masaki A. Ito, Poorva Sandlesh, Imon Goswami, Laura A. Prendergast, Katerina Gurova. Conditional knockout of histone chaperone FACT in mice: Consequences for normal tissues [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 5193.
Abstract 5202: Molecular mechanism of transcriptional regulation of REV7, a DNA damage response gene
Authors: Murakumo Yoshiki , Shimada Yuko , Kato Takuya , Sakurai Yasutaka
Published: August 2019

REV7 is involved in several biological response after DNA damage, including the double strand break repair and translesion DNA synthesis. It has bee reported that high levels of REV7 expression are observed in several malignant tumors and are associated with resistance to chemotherapeutic agents such as cisplatin. In normal tissues, REV7 expression is extremely high in germ cells compared with the other normal cells, suggesting the cell type-specific expression of REV7. However, the mechanism of regulation of REV7 expression has not been understood. To assess the mechanism of regulation of REV7 expression, we analyzed the promotor region of the human REV7 locus to identify transcriptional regulators controlling REV7 expression. The transcriptional start point of the REV7 gene was determined by 5’-RACE using human testis cDNA. About 3 kb of the upstream region of the REV7 locus was isolated by genomic PCR and the promoter activity was analyzed by the luciferase reporter assay. Using several deletion mutants, we found that there are two transcriptional activation regions in the upstream of the REV7 locus. Bioinformatic analyses revealed several motifs of transcription factor binding sites in these regions, and we focused on one molecule as a candidate for the transcription factor controlling REV7 expression. We will present and discuss our further analyses.

Note: This abstract was not presented at the meeting.

Citation Format: Yoshiki Murakumo, Yuko Shimada, Takuya Kato, Yasutaka Sakurai. Molecular mechanism of transcriptional regulation of REV7, a DNA damage response gene [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 5202.
Abstract 5217: RB loss reprograms AR and E2F1 signaling in models of prostate cancer progression
Authors: Mandigo Amy C. , McNair Chris , Schiewer Matthew J. , Knudsen Karen E.
Published: August 2019

Prostatic adenocarcinoma (PCa) is the most frequently diagnosed non-cutaneous malignancy, and the third leading cause of cancer death in males in the United States. A crucial component to the development and progression of PCa is the activity of the androgen receptor (AR). As such, targeting the AR-signaling axis through androgen deprivation therapy (ADT) is the first line of therapy for PCa. However, cells invariably become resistant to this therapy and men relapse with the incurable form of the disease termed, castration-resistant prostate cancer (CRPC). In addition to AR, another principal component aiding in the progression of disease is the retinoblastoma tumor suppressor protein (RB). RB functions to repress tumor development by negatively regulating the activity of the E2F family of transcription factors, preventing cell cycle progression. RB is lost in roughly 30% of CRPC tumors and is sufficient to induce a CRPC phenotype in hormone-sensitive cells under ADT conditions. Analyses into the molecular significance of RB loss on disease progression identified a potential cooperation between AR and the RB-E2F1 signaling axis. Biological assessment performed in isogenic RB knockdown (i.e. hormone-sensitive and CRPC models) identified distinct functional consequences of RB loss depending on AR status and disease state. Transcriptome analysis identified divergently regulated gene signatures between disease stages in the presence of AR activation, which were not seen under ADT conditions, implicating a unique role of AR in transcriptional regulation with the loss of RB. Data to be discussed will also include further comparison of the E2F1 and AR cistromes in the absence of RB to identify the mechanism by which the AR-RB-E2F1 signaling axis function in promoting the progression of disease.

Citation Format: Amy C. Mandigo, Chris McNair, Matthew J. Schiewer, Karen E. Knudsen. RB loss reprograms AR and E2F1 signaling in models of prostate cancer progression [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 5217.
Abstract 5226: Non-canonical functions of TERT in glioblastoma pathobiology
Authors: Ferdosi Shayesteh R. , Bollam Saumya , Peng Sen , Taylor Brett , Gokhale Vijay , Hurley Laurence , Berens Michael , Dhruv Harshil
Published: August 2019

Mutations in the promotor region of TERT are the most common non-coding mutations across cancers, especially in glioblastoma (GBM), which has a TERT mutation frequency rate over 80%. In the absence of any effective molecular targeting therapy for GBM, elucidating oncogenic signaling of TERT could open new avenues in GBM treatment. Canonically, mutations of TERT, which result in TERT upregulation, maintain telomere length in the nucleus and promote indefinite proliferation of cancer cells. However, a non-canonical function of TERT in the mitochondria has recently been suggested. We screened GBM cell models against a novel small molecule inhibitor (RG1534, Reglagene Inc.) that interferes with the functionality of a mutated hTERT promoter. RG1534 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes. More interestingly, RG1534 treatment leads to rapid apoptosis induction in glioma cell lines that does not correlate with the time course of the telomere shortening effect. We further validated this rapid apoptosis behavior in glioma cell lines using siRNA and CRISPR/Cas-9 mediated hTERT knockdown. We also measured the protein expression of TERT in subcellular fractions of glioma cell lines and demonstrated the presence of higher TERT expression in mitochondrial extract compared to the nuclear extract. Finally, using MitoSOX dye we demonstrated significant increase of ROS generation in glioma cells treated with RG1534 as compared to control. In summary, our results demonstrate that non-canonical functions of TERT may play critical role in glioma pathobiology and require more detail investigation.

Citation Format: Shayesteh R. Ferdosi, Saumya Bollam, Sen Peng, Brett Taylor, Vijay Gokhale, Laurence Hurley, Michael Berens, Harshil Dhruv. Non-canonical functions of TERT in glioblastoma pathobiology [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 5226.
Abstract 5234: Epigenetic control of heparanase expression through CRISPR/dCas9
Authors: Zeng Guihua , Liang Fu-Sen , Cui Lina
Published: August 2019

Heparanase is the only known enzyme that cleaves heparan sulfate (HS) and hence participates in the degradation and remodeling of the extracellular matrix (ECM). Pathological expression of heparanase at abnormal levels can contribute to the development of various diseases. In our research, directed by clustered regularly interspaced short palindromic repeats (CRISPR) system, the promoter region of heparanase was successfully and specifically targeted with screened and combined single guide RNAs (sgRNAs). Linking dCas9 with different histone modification enzymes (eg. P300, hHDAC3, LSD1), the expression of heparanase was successfully up- or down-regulated through acetylation, deacetylation and demethylation. This approach allows us to tune heparanase expression in various cell lines on demand, enabling the study of heparanase activity in real time.

Citation Format: Guihua Zeng, Fu-Sen Liang, Lina Cui. Epigenetic control of heparanase expression through CRISPR/dCas9 [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 5234.
Abstract 5241: Whole transcriptome TempO-Seq profiling of focal areas of H&E stained FFPE: Differentiation of normal colon and cancer phenotypes between donors
Authors: Trejo Christy , Imler Elliot , Babic Milos , Shepard Peter , Nagle Raymond , Yeakley Joanne , Seligmann Bruce
Published: August 2019

Profiling gene expression from small amounts of FFPE using methods that require extraction of RNA from the tissue, such as RNA-seq, have been problematic. We report use of a modification of the whole transcriptome TempO-Seq® assay (Yeakley et al, PLOSone, 2017; DOI:10.1371/journal.pone.0178302) to profile 2 mm2 areas of 5 μm thick FFPE sections by scraping the tissue and assaying lysates directly without extracting the RNA. Duration of fixation and the amount of sample down to a 2 mm2 area do not impact the performance of the assay. Good results can be achieved using FFPE blocks that are up to 30 years old. As a consequence of the ability to assay areas as small as 2 mm2 from H&E stained tissue, we were able to scrape and profile areas of colorectal cancer vs adjacent normal and obtain replicates within donors, and then compare between donors. The assay shows excellent reproducibility, with R2 > 0.95 between biological replicates (separate samples taken from the same patient and lysed/processed separately). While the normal tissues from the patients exhibited a range of expression variability, they all clustered very tightly when analyzed via principal component analysis. The cancer tissue profile from each patient was not only substantially different from the normal tissue profile of that patient, but substantially different from all profiles within the normal tissue cluster of all patients. Furthermore, the cancer tissue profiles were separated into distinct subgroups. Changes in regulatory pathways leading to oncogenesis could be directly distinguished between patients. These results demonstrate the ability to use whole transcriptome TempO-Seq profiling to investigate molecular subtypes of colon cancer using archival FFPE samples. We are expanding the cohort of patients being profiled to investigate these signatures further, and to draw out signatures which allow direct subtype classification purely from expression data.

Citation Format: Christy Trejo, Elliot Imler, Milos Babic, Peter Shepard, Raymond Nagle, Joanne Yeakley, Bruce Seligmann. Whole transcriptome TempO-Seq profiling of focal areas of H&E stained FFPE: Differentiation of normal colon and cancer phenotypes between donors [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 5241.
Abstract 5245: Integrative analysis of KIF4A, 9, 18A, and 23 and their clinical significance in low-grade glioma and glioblastoma multiforme
Authors: Cho SangYeon , Kim DongWoon
Published: August 2019

To determine the prognostic significance of kinesin superfamily gene (KIF) expression in patients with brain cancer, including low-grade glioma (LGG) and glioblastoma multiforme (GBM), we comprehensively analyzed KIFs in 515 LGG and 595 GBM patients. Among KIFs, KIF4A, 9, 18A, and 23 showed significant clinical implications in both LGG and GBM. The mRNA and protein expression levels of KIF4A, 9, 18A, and 23 were significantly increased in LGG and GBM compared with those in the normal control groups. The mRNA expression levels of KIF4A, 9, 18A, and 23 in LGG were significantly increased in the high-histologic-grade group compared with those with a low histologic grade. Genomic analysis showed that the percent of mRNA upregulation of KIF4, 9, 18A, and 23 was higher than that of other gene alterations, including gene amplification, deep deletion, and missense mutation. In addition, LGG patients with KIF4A, 18A, and 23 gene alterations were significantly associated with a poor prognosis. In survival analysis, the group with high expression of KIF4A, 9, 18A, and 23 mRNA was significantly associated with a poor prognosis in both LGG and GBM patients. Gene Set Enrichment Analysis (GSEA) revealed that high mRNA expression of KIF4A, 18A, and 23 in LGG and GBM patients showed significant positive correlations with the cell cycle, E2F targets, G2M checkpoint, Myc target, and mitotic spindle. By contrast, high mRNA expression of KIF9 in both LGG and GBM patients was significantly negatively correlated with the cell cycle, G2M checkpoint, and mitotic spindle pathway. However, it was significantly positively correlated with EMT and angiogenesis. This study has extended our knowledge of KIF4A, 9, 18A, and 23 in LGG and GBM and shed light on their clinical relevance, which should help to improve the treatment and prognosis of LGG and GBM.

Citation Format: SangYeon Cho, DongWoon Kim. Integrative analysis of KIF4A, 9, 18A, and 23 and their clinical significance in low-grade glioma and glioblastoma multiforme [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 5245.
Abstract 5270: A potential role of branched chain amino acid metabolism in breast cancer cell invasiveness
Authors: Colonna Maxwell B. , Edison Arthur S. , Zhao Shaying
Published: August 2019

Carcinomas have two broadly distinct modes of invasion: collective and individual. Collective invasion is characterized as groups of cells that migrate while retaining cell-cell contacts. Individual invasion occurs through mechanisms such as the acquiring of mesenchymal-like features by cancer cells. While these different modes of invasion have been well studied at the genomic and transcriptional levels, their metabolic alterations remain much less understood. To address this deficiency, we performed untargeted NMR metabolomics, in conjunction with RNA-seq, on two variants of MCF7, a widely used breast cancer cell line. We report herein several findings. First, the two MCF7 variants, one parental (referred to as MCF-7 hereafter) and another having undergone many passages (referred to as Augusta hereafter), differ morphologically, with MCF-7 being more epithelial and Augusta being more mesenchymal-like. Importantly, fibroblast co-culture experiments indicate that MCF-7 cells resemble collective invasion, while Augusta cells resemble individual invasion. Furthermore, both scratch-assay and gene set enrichment analysis of RNA-seq data indicate that Augusta cells are more invasive. Second, our NMR metabolomics analysis reveals that branched chain amino acids (BCAAs) represent a significant metabolic difference between MCF-7 and Augusta cells. Partial least squares discriminant analysis of the metabolomics data identified BCAAs as essential distinguishing features between the two cell lines. Specifically, compared to MCF-7 cells, BCAAs are significantly depleted inside Augusta cells, consistent with the upregulation of several key BCAA degradation genes. We hypothesize that Augusta cells can more efficiently utilize BCAAs, and are performing 13C-labeled BCAA flux analysis to test this hypothesis. Previous studies have shown that an aberrantly expressed BCAA metabolism gene sustains growth and enhances invasiveness of breast cancer cells. However, increased catabolism of BCAAs has not been reported in literature on this subject. Our simple cell line model allows for a deeper molecular understanding of BCAA metabolism in breast cancer cell invasion.

Citation Format: Maxwell B. Colonna, Arthur S. Edison, Shaying Zhao. A potential role of branched chain amino acid metabolism in breast cancer cell invasiveness [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 5270.
Abstract 5273: Multi-omics approaches reveal potential role for corticosterone in prostate cancer
Authors: Mahmud Iqbal , Lee Bongyong , Perera Ranjan , Garrett Timothy J.
Published: August 2019

Prostate cancer (PCa) is the most commonly diagnosed cancer in American men. In 2018, 164,690 new cancer cases and 29,430 cancer deaths are projected to occur in the United States. Currently, there is no efficient diagnostic and safe treatment option for the management of PCa. Using Ultra-High Performance Liquid Chromatography coupled High Resolution Mass Spectrometry (UHPLC-HRMS), we globally profiled more than 1400 metabolites across 79 urine samples related to PCa (19 control, 23 benign prostate hyperplasia (BPH), 11 prostatitis, and 26 PCa samples). Among all, corticosterone was identified as a top differential metabolite. Corticosterone levels were markedly elevated in PCa urine samples and modest but significant elevation of the corticosterone in BPH and prostatitis urine samples compared to controls, with an excellent area under the receiver operating characteristic curve of 0.95, 0.94, and 0.93, respectively. In addition, the expression of corticosterone biosynthetic gene, Steroid 11-beta-hydroxylase (CYP11B1), was elevated in PCa tissues, cell lines, and urine samples, while Corticosteroid 11-beta-dehydrogenase isozyme 2 (HSD11B2), which metabolize corticosterone, were markedly reduced in prostate tumors. With the understanding that androgen signaling is the key factor for prostate cancer, we investigated the role of androgen in regulating CYP11B1 and HSD11B2. We analyzed several androgen treated PCa cell lines derived global gene expression datasets and revealed that treatment with androgen increase in CYP11B1 expression and a concomitant decrease in HSD11B2 expression levels. Interestingly, we uncover that genetic knockdown of Androgen Receptor (AR) diminished CYP11B1 expression and alternatively, increased HSD11B2. Moreover, AR based chromatin occupancy (ChIP-seq) data also suggest direct binding of AR to the promoter of CYP11B1 in multiple PCa cells. The role of corticosterone in PCa is previously unknown. Our multi-omics approaches thus indicate that corticosterone might be potential prognostic marker for PCa. The tumor biology and mechanism through which corticosterone associates with PCa is being investigated in our laboratory.

Citation Format: Iqbal Mahmud, Bongyong Lee, Ranjan Perera, Timothy J. Garrett. Multi-omics approaches reveal potential role for corticosterone in prostate 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 5273.
Abstract 538: LC-MS based catabolite identification study of an ADC with DM21-C, a novel maytansinoid linker-payload
Authors: Lau Janet , Salomon Paulin , Sinkevicius Kerstin , Costoplus Juliet , Fuller Megan , Xu Raymond , Hicks Stuart , Chari Ravi , Widdison Wayne , Yoder Nicholas , Keating Thomas
Published: August 2019

ImmunoGen’s newest antibody-drug conjugate (ADC) design uses the novel maytansinoid linker-payload, DM21-C that bears a peptidase/protease cleavable linker. These ADCs show good bystander killing of proximal antigen negative cells, suggesting the generation of cell-permeable catabolites. The goal of this study was to identify the catabolites generated upon incubation in antigen-positive cancer cells (both cell pellet and media), in mouse plasma, as well as in in vitro catabolic systems. Upon incubation with a cysteine-conjugated DM21-C ADC, the small molecule fraction was extracted with an organic solvent and analyzed by high resolution mass spectrometry after chromatographic separation. Through comparison to reference standards, mass spectral signal extraction of possible products, and database search of unknown peaks, the potential in vitro catabolite products of DM21-C conjugate were identified. Information on linker-payload stability in these model systems, including plasma stability, in vitro metabolism of the ADC, and catabolic products that were both retained and effluxed from cancer cells was obtained. As a result, we identified DM51 (the thiol- resulting from self-immolation of the cleaved linker-payload) as a major catabolite of the DM21-C ADC.

Citation Format: Janet Lau, Paulin Salomon, Kerstin Sinkevicius, Juliet Costoplus, Megan Fuller, Raymond Xu, Stuart Hicks, Ravi Chari, Wayne Widdison, Nicholas Yoder, Thomas Keating. LC-MS based catabolite identification study of an ADC with DM21-C, a novel maytansinoid linker-payload [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 538.
Abstract 543: Dinutuximab binds specifically to disialoganglioside-2
Authors: Yaghoubi Shahriar , Harrison Travis , Messerschmidt Gerald , Corritori Suzana
Published: August 2019

Dinutuximab is a human/murine chimeric monoclonal antibody, approved for maintenance treatment of pediatric patients with high-risk neuroblastoma (NB) in the US, and currently under investigation in a phase III trial (NCT03098030) for 2nd line treatment of relapsed/refractory small cell lung cancer (SCLC). Despite four decades of development, the specificity of dinutuximab binding to its intended target, disialoganglioside-2 (GD2), has not been fully elucidated. Although GD2 is a relatively cancer cell specific antigen, other cell surface gangliosides are ubiquitously expressed on the outer membrane of healthy human cells. Therefore, the main goal of this study was to confirm binding specificity of dinutuximab to GD2. We also compared relative affinity of dinutuximab to GD2 with 14G2a, which is the original murine anti-GD2 antibody from which dinutuximab was derived. To measure dinutuximab and 14G2a binding kinetics to various relevant gangliosides (GD1a, GD1b, GM3, GD3, GM4, GM2 and GD2), an in vitro assay was developed and optimized using the Biacore surface plasmon resonance platform. The results showed that GD2 was the only ganglioside that binds to dinutuximab (relative KD=0.000337M). The relative affinity of GD2 to dinutuximab and 14G2a antibody were comparable and within 2-fold of each other (Table). In conclusion, dinutuximab specifically binds to GD2. The absence of binding to GM3, GD3, GD1a, or GD1b suggests that an exposed N-acetylgalactosamine (GalNAc) moiety is necessary for antibody recognition. The absence of binding to GM2 or GT2 (sugar moiety), which differ from GD2 by the number of sialic residues attached to the basal galactose moiety, suggests that the number of sialic acid residues is also critical for antibody recognition.

GD2 binding kinetics of Dinutuximab and 14G2aLigandKD (M)ka (1/Ms)kd (1/s)Chi2 (RU2)Dinutuximab3.37E-042.48E+028.34E-020.78614G2a2.00E-047.11E+021.42E-010.0273

Citation Format: Shahriar Yaghoubi, Travis Harrison, Gerald Messerschmidt, Suzana Corritori. Dinutuximab binds specifically to disialoganglioside-2 [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 543.
Abstract 545: Preclinical development of a bispecific antibody-trap selectively targeting CD47 and CD20 for the treatment of B cell lineage cancer
Authors: Tian Wenzhi , Li Song , Chen Dianze , Liang Guangjun , Zhang Li , Zhang Wei , Tu Xiaoping , Peng Liang , Weng Jie , Zhao Gui , Liu Dandan , Guo Huiqin , Yang Chunmei , Zhang Ruliang
Published: August 2019

Tumor cells over expressing CD47 can evade immune surveillance through delivering a “Don’t eat me” signal to macrophages. Developing CD47-targeted modalities has been a hot pursue in the pharmaceutical industry. However due to the ubiquitous expression of CD47 in normal tissues, especially in red blood cells (RBC), severe antigenic sink as well as hemotoxicity including anemia set up a higher bar for this particular targeted therapy development. To overcome these hurdles, we have developed a bispecific recombinant antibody-trap, IMM0306, designed to simultaneously target CD47 and CD20 on B cells but avoid binding to human RBCs. Extensive in vitro characterization demonstrated that IMM0306 binds to both CD47 and CD20 with affinity 3-8 folds lower than either single-targeted molecule, but has strong pro-phagocytosis activity over CD47-positive target cells, and even stronger ADCC activity than Rituximab. Intriguingly, IMM0306 has no binding activity at all toward human RBCs, albeit much lower binding activity toward monkey RBCs. Treatment of tumor-implanted SCID mice with IMM0306 significantly inhibited tumor growth and led to eradication of the tumor cells from 5 out of 8 mice, which is much more effective than Rituximab and than co-administration of Rituximab and SIRPα-Fc (IMM01), although profound synergistic effect has been observed for the co-administration. Preclinical study in non-human primates demonstrated a favorable pharmacokinetic profile with no obvious hemotoxicity following single as well as multiple administrations at different dosage. Our study suggests that antibody-trap like IMM0306 might be an ideal approach for CD47-targeted immunotherapy development since selective avoidance of RBC mediated antigen-sink as well as anemia could be achieved along with the robust anti-tumor activity.

Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Guangjun Liang, Li Zhang, Wei Zhang, Xiaoping Tu, Liang Peng, Jie Weng, Gui Zhao, Dandan Liu, Huiqin Guo, Chunmei Yang, Ruliang Zhang. Preclinical development of a bispecific antibody-trap selectively targeting CD47 and CD20 for the treatment of B cell lineage 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 545.
Abstract 61: ECM scaffolds to elucidate the role of Col11a1 in head and neck cancer progression
Authors: Thomas Sufi M. , Enders Jonathan , Isai Greta , Czirok Andras
Published: August 2019

Cancer progression is linked to physical properties, cellular and biochemical composition of the tissue environment including the extracellular matrix (ECM). The ECM not only provides signaling cues by presenting ligands, but also plays a key role in mechano-sensing: modulation of biochemical pathways by physical forces. Collagens are one of the most abundant components of the ECM. We previously reported that head and neck squamous cell carcinoma (HNSCC), which is the 6th most common cause of cancer mortality worldwide with < 40% 5-year survival rates, express higher levels of collagen 11a1 (Col11a1) than normal adjacent mucosa. Col11a1 is a minor fibrillar collagen whose main physiologic role is to regulate the diameter of major collagen fibrils in the cartilage. Although reported that Col11a1 facilitates HNSCC proliferation and invasion, not much is known about its role in the tumor microenvironment. We tested the hypothesis that Col11a1-cancer interdependence facilitates tumor adhesion and metastatic colonization. We developed ECM substrates from cell lines and HNSCC tumor tissue that were characterized by scanning electron microscopy and immunofluorescence microscopy. Electrospun meshes with ECM substrates were developed and used to assess HNSCC adhesion, proliferation and motility. We used a 3D multi-cell type spheroid culture model called the primitive-lung-in-a-dish (PLiD) to demonstrate that Col11a1 siRNA targeting attenuates HNSCC colonization in the lung. Further, using homogenized and lyophilized decellularized ECM from mouse lungs, we demonstrate that Col11a1 regulates motility of HNSCC. Overall, we have used several models to elucidate the role of Col11a1 in HNSCC adhesion and metastatic colonization.

Citation Format: Sufi M. Thomas, Jonathan Enders, Greta Isai, Andras Czirok. ECM scaffolds to elucidate the role of Col11a1 in head and neck cancer progression [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 61.