bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4363: A-FABP and estrogens are independently involved in the development of breast cancer
Authors: Li Bing , Hao Jiaqing , Yan Xiaofang , Kong Maiying , Sauter Edward
Published: September 2020

Background: We previously reported that postmenopausal obese women exhibit increased levels of circulating adipocyte fatty acid binding protein (A-FABP), which is associated with breast cancer (BC) development. In postmenopause, increased estrogen levels are associated with increased BC risk.

Hypothesis: Estrogens (estrone: E1, estradiol: E2 and estriol: E3) are associated with A-FABP in the setting of obesity and obesity-related BC development.

Methods: Immunoassays for E1, E2, E3, and A-FABP were performed using serum samples from women with and without BC.

Results: Statistical analysis was conducted using the Mann-Whitney test and multiple linear regression models. Considering all subjects, E1 and E2 but not E3 levels were significantly higher in pre- than in postmenopause among both non-obese and obese individuals (p<0001). E3 (p<0.01) and E1 (p<0.05) levels were higher in non-obese than in obese women. A-FABP levels were significantly higher in obese postmenopausal women with and without BC, but not in premenopausal women, regardless of BC status. When stratified by BC status, E2 levels were significantly higher, while E1 and E3 levels were significantly lower in postmenopausal healthy obese than non-obese women. These body mass index (BMI) based differences were not observed among women with BC. E3 levels were higher in obese pre (p=0.003) and postmenopausal (p=0.001) women with BC than those without. A-FABP levels were significantly higher in postmenopausal obese healthy women and women with BC. A-FABP was not highly associated with E1, E2 or E3.

Impact: 1) A-FABP expression is increased in postmenopausal obese women with and without BC; 2) E1 and E2 expression is higher in pre- than postmenopausal women, regardless of BMI; 3) E1 and E3 levels are higher in lean than obese individuals, regardless of menopausal status; 4) the influence of obesity on E1, E2 and E3 expression in postmenopause is lost in women with BC, 5) E3 is increased in obese vs. non-obese women with BC, and 6) A-FABP and estrogens are independently involved in the development of BC.

Citation Format: Bing Li, Jiaqing Hao, Xiaofang Yan, Maiying Kong, Edward Sauter. A-FABP and estrogens are independently involved in the development of breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4363.
Abstract 4370: Lasofoxifene alone or in combination with palbociclib as an effective treatment for therapy-resistant ER-positive metastatic breast cancer
Authors: Laine Muriel , Kurleto Justyna D. , Greene Marianne E. , Phung Linda , Komm Barry S. , Greene Geoffrey L.
Published: September 2020

Patients with Estrogen Receptor positive (ER+) metastatic breast cancer (MBC) are currently treated with fulvestrant, a selective estrogen receptor degrader (SERD), or a combination of fulvestrant and palbociclib, a CDK4/6 inhibitor. Approximately 25-40% of patients with MBC have activating mutations in the ERα ligand-binding domain. The two most common mutations are Y537S and D538G, which confer constitutive activity to ERα, resulting in AI/tamoxifen/fulvestrant resistance. Lasofoxifene, a selective estrogen receptor modulator (SERM), was developed for the treatment of vaginal atrophy and osteoporosis. Previous studies from this lab, performed in a mutant ERα MCF-7 xenograph MBC mouse model, showed that lasofoxifene was significantly more effective than fulvestrant at inhibiting tumor growth, as well as metastasis to the lung and liver. In the current dose/response study, we compared the efficacy of lasofoxifene + palbociclib combinations to fulvestrant + palbociclib combinations. Engineered and luciferase-GFP tagged MCF-7 Y537S cells were injected into the mammary ducts of NSG mice (MIND model) and tumor progression was monitored by live luminescence imaging of primary tumors as well as ex vivo imaging of metastatic sites at study endpoint. Our results showed that all dose combinations of lasofoxifene + palbociclib were more effective than fulvestrant + palbociclib at inhibiting primary tumor growth as well as bone, lung, liver and brain metastasis. These data strongly suggest that patients with ERα+ MBC would benefit from orally delivered lasofoxifene + palbociclib therapy and that this combination could be superior to fulvestrant + palbociclib.

Citation Format: Muriel Laine, Justyna D. Kurleto, Marianne E. Greene, Linda Phung, Barry S. Komm, Geoffrey L. Greene. Lasofoxifene alone or in combination with palbociclib as an effective treatment for therapy-resistant ER-positive metastatic breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4370.
Abstract 4374: CE+BZA combination therapy decreases breast cancer outgrowth in part through activation of the AHR pathway
Authors: Dembo Anna G. , Aledort Emily S. , Greene Geoffrey L.
Published: September 2020

Menopause occurs in all women, usually between the ages of 45 and 55. As a result of menopause, women often experience undesirable vasomotor symptoms, which can be alleviated by hormone replacement therapy (HRT). The Women's Health Initiative (WHI) trial concluded that PremPro, a HRT formulation that combines conjugated equine estrogens (CE) with medroxyprogesterone acetate, increases the risk of breast cancer. Over the years, the number of women taking HRT has dramatically decreased due to the perceived risk based largely on the results of the WHI trial. Follow-up studies suggest that breast cancer cases from PremPro treatment were primarily due to the outgrowth of occult tumors, not the formation of new disease. Duavee, a more recent form of HRT that combines CE and bazedoxifene (BZA), a selective estrogen receptor modulator (SERM) and degrader (SERD), has been approved by the FDA for treatment of moderate to severe hot flashes and to reduce the risk of osteoporosis. More importantly, this CE+BZA mixture not only relieves symptoms associated with menopause, but it also does not stimulate the breast or uterus. Several preclinical studies suggest that CE+BZA might be protective in the breast, however the mechanism of action of this new combination therapy is not known. Our goal, therefore, was to elucidate the underlying molecular mechanisms by which CE+BZA differentially affects estrogen receptor alpha (ERα) action in the mammary gland. Pathway analysis from RNA-sequencing revealed Aryl Hydrocarbon Receptor (AHR) signaling as the top canonical pathway affected by treatment with CE+BZA. Previous studies reported that 4-hydroxytamoxifen, a metabolite of the SERM tamoxifen, was able to activate the AHR pathway. In our study we determined that BZA treatment activated the AHR pathway in both ERα-positive and ERα-negative cell lines. BZA increased expression of aryl hydrocarbon receptor repressor (AHRR) in cell lines and increased expression of two AHR pathway members, CYP1A1 and CYP1B1, in tumors of estrogen-sensitive polyoma middle T antigen (PyMT) mice. AHRR has been reported to act as a tumor suppressor in breast cancer; therefore activation of AHR by BZA in women taking CE+BZA combination therapy might be one mechanism by which outgrowth of occult tumors is decreased. Further evaluation of the role of AHR in breast cancer is necessary to improve our understanding of CE+BZA action.

Citation Format: Anna G. Dembo, Emily S. Aledort, Geoffrey L. Greene. CE+BZA combination therapy decreases breast cancer outgrowth in part through activation of the AHR pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4374.
Abstract 4375: Inhibition of triple negative breast cancer cell progression using estrogen related receptor alpha endogenous ligands
Authors: Ghanbari Faegheh , Mader Sylvie , Philip Anie
Published: September 2020

Triple-negative breast cancer (TNBC), which lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), is considered to be more metastatic, and has poorer prognosis and higher risk of recurrence than other subtypes of breast cancer. The death rate in patients with TNBC is twice that of ERα positive tumors mainly because there are fewer targeted therapies that treat TNBC patients [1]. Therefore, there is a need to discover new drug-targeted therapy for these patients. Several lines of evidence indicate that estrogen related receptors (ERRs), which belong to the orphan nuclear receptor superfamily, play a crucial role in breast cancer, with ERRα overexpression reportedly leading to adverse clinical outcomes in TNBC patients [2]. Despite intensive efforts, no endogenous ligand has been identified for ERRs so far. The discovery of ligands that bind these receptors may lead to novel strategies for the treatment of TNBC. In this study we show two endogenous ligands of ERRs: 1) estradienolone (ED), a novel endogenous steroid during pregnancy which acts as an inverse agonist of ERRs, 2) cholesterol as an agonist of ERRs. Our recent results show that ED acts as an inverse agonist of ERRα and ERRγ by directly interacting with these receptors, and inhibiting their transcriptional activity. We also demonstrate that ED has strong anti-mitogenic properties. ED inhibits the growth of both estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) breast cancer cells in a dose dependent manner, while of displaying a little effect on normal epithelial breast cells. In addition, we show that cholesterol binds directly and specifically to ERRα, it increases the transcriptional activity of ERRα in a peroxisome proliferator-activated receptor coactivator-1α (PGC-1) dependent manner. Our finding suggests that cholesterol enhances the interaction of ERRα and its coactivator PGC-1, and this leads to induce the expression of ERRα itself (an specific auto-induction) and the metabolic target genes of ERRα to fuel TNBC cells proliferation and migration. Moreover, our results demonstrates that the effect of cholesterol or lovastatin (a drug known to inhibit cholesterol synthesis) in triple negative breast cancer (MDA-MB 231) requires ERRα. These data suggest that both ED-ERR and cholesterol-ERR interactions may represent novel druggable signaling pathway in TNBC.

Citation Format: Faegheh Ghanbari, Sylvie Mader, Anie Philip. Inhibition of triple negative breast cancer cell progression using estrogen related receptor alpha endogenous ligands [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4375.
Abstract 4378: Contribution of ERα/PR crosstalk to endocrine therapy resistance in breast cancer
Authors: Huggins Rosemary J.
Published: September 2020

Half of estrogen receptor (ERα)-positive breast cancer patients treated with endocrine therapies display intrinsic or acquired therapy resistance. One-third of patients with acquired resistance present with metastatic tumors containing ERα-Y537S mutations. This mutation results in constitutive activity of ERα and altered ERα-associated gene expression. ERα/PR crosstalk, referring to the complex relationship of ERα and progesterone receptor (PR) interaction, offers therapeutic value in targeting both nuclear receptors simultaneously in ERα/PR-positive breast cancers. Previous research identified a synergistic effect of treating ERα-WT cancer cells with ERα/PR antagonism, where combined treatment results in tumor regression in vivo and increased cell death in vitro. Conversely, preliminary data suggests that treatment of ERα-Y537S-containing tumors with combined ERα and PR antagonists results in significantly increased proliferation in vivo and reduced cell death in vitro. My central hypothesis is that ERα-Y537S alters the transcription factor activity of ERα and PR, causing dysregulation of gene expression in SERM-resistant breast cancer. Using a three-dimensional culture system of patient-derived xenografts grown as organoids (PDxO), I have observed a synergistic effect of certain combinations of ERα/PR antagonists in the context of ERα-WT similar to previous results in two-dimensional cell culture. At the level of transcription, PR-associated transcriptional activity appears to be significantly altered in homozygous ERα-Y537S T47D cells compared to ERα-WT cells. Further understanding the role of ERα-Y537S in altering gene expression and reducing the response to SERM and SPRM treatment will provide understanding as to why these tumors are resistant to standard-of-care treatment and will suggest alternative targets for treatment as well.

Citation Format: Rosemary J. Huggins. Contribution of ERα/PR crosstalk to endocrine therapy resistance in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4378.
Abstract 4382: Bioinformatics analysis for hub genes and pathways in hepatocellular carcinoma
Authors: Zeng Xianzhang , Ao Lu , Lin Xinjian , Lin Xu , Chen Wannan
Published: September 2020

Hepatocellular carcinoma (HCC) is the most common primary liver cancer with a high recurrence rate and mortality. There is no effective treatment for HCC and the molecular mechanisms of HCC are still not fully understood. To identify the potential biomarkers in the carcinogenesis and progression of HCC, microarray datasets GSE101685, GSE121248, GSE38941, GSE62232 and GSE87630 were downloaded from Gene Expression Omnibus (GEO) database. Screening of differential expressed genes (DEGs), and functional enrichment analyses were performed. The protein-protein interaction network (PPI) were constructed and significant module genes and hub genes were identified using STRING and Cytoscape. 149 DEGs were screened, including 114 down-regulated genes and 35 up-regulated genes. Fifteen hub genes were identified and biological process analysis revealed that these genes were mainly enriched in cell cycle, oocyte meiosis and protein binding. Survival analysis and co-expression network analysis showed that TOP2A, TPX2, RACGAP1 and PTTG1 may be involved in the carcinogenesis, invasion or recurrence of HCC. In conclusion, the present study may help us understand the molecular mechanisms underlying the carcinogenesis and progression of HCC, and provide candidate targets for diagnosis and treatment of HCC.

Key words: Hepatocellular carcinoma; Differentially expressed genes; Protein-protein interaction; Bioinformatics; Microarray

Citation Format: Xianzhang Zeng, Lu Ao, Xinjian Lin, Xu Lin, Wannan Chen. Bioinformatics analysis for hub genes and pathways in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4382.
Abstract 4388: Weighted gene co-expression network analysis identified cell cycle signaling pathway associated hub genes correlated with progression and prognosis of multiple myeloma
Authors: Adebayo Olayinka O. , Griffen Tiara , Young Corey , Dammer Eric , Lillard James W.
Published: September 2020

There is a gap in understanding the molecular mechanisms behind early relapse / multiple myeloma (MM) progression in the newly diagnosed patient on standard treatment for MM, i.e., lenalidomide, bortezomib, and dexamethasone (RVD). As a result, the relatively high percentage of patients that progress after early treatment with RVD is significant. Therefore, identification of clinically relevant clusters of co-expressed genes or representative biomarkers for MM progression, while on RVD, would help identify new molecular mechanisms and drug targets. The objective of this study is to use weighted gene co-expression network analysis (WGCNA) to identify gene-signaling networks associated with early relapse / MM progression. To this end, we performed a WGCNA to determine module-trait correlations. We next examined the overrepresentation of upstream regulators and signal pathway networks from MM patients in the MMRF CoMMpass dataset (n = 175, gene transcripts= 30,598). WGCNA constructed 48 modules based on the correlations between patients on RVD and death. We are using death as a biomarker for MM progression and two years as cut off for treatment. We identified two modules, Green (p < 4.6 x 10-8), and Pale Turquoise (p < 8.8 x 10-7), that significantly and positively correlated with overall survival of < 2 years following initial treatment with RVD. Further analysis identified HDAC8, PARPBP, HSPG2, MAGE, KIF, and FOXM1 as the top six hub genes having tight variations in positive correlations. The signatures for these cell cycle signaling pathway related hub genes were found to be largely associated with the biological process for proliferation. Future studies will use connectivity mapping to identify drug signatures that can target any of the identified hub genes. The selective targeting of these hub genes is expected to improve response to RVD in MM patients. This transcriptome study is the first using this approach to identify gene signaling networks associated with MM progression.

Citation Format: Olayinka O. Adebayo, Tiara Griffen, Corey Young, Eric Dammer, James W. Lillard. Weighted gene co-expression network analysis identified cell cycle signaling pathway associated hub genes correlated with progression and prognosis of multiple myeloma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4388.
Abstract 4389: Glycogen synthase kinase 3A (GSK3A) gene alterations are negatively correlated with overall and disease-free survival in breast cancer patients: In silico analysis
Authors: Kaur Jasmeet , Kaur Karandeep
Published: September 2020

Breast cancer is a heterogeneous disease and most widespread malignancy in women. It is one of three leading causes of deaths worldwide, together with lung and colon cancer. Glycogen synthase kinase 3 (GSK3) is a serine/threonine protein kinase involved in glucose regulation, inflammation, immune responses, proliferation, migration and apoptosis. GSK3 is expressed in all mammalian tissues and has a role in diseases including mood disorders, Alzheimer disease, diabetes and cancer. GSK3A and GSK3B are two isoforms, encoded by distinct genes and are 98% identical within their kinase domain. Gene knockout studies demonstrate that the two isoforms are not completely redundant, but perform similar functions in several settings. So far, very little is known about GSK3A role in cancer. We analyzed correlation of GSK3A gene mutations and copy number changes with overall and disease-free survival in breast cancer patients using cBioPortal for cancer genomics. Fourteen datasets for breast cancer studies were analyzed and GSK3A alterations were found in seven datasets. These alterations included mutations (missense and truncating), amplifications and deep deletions; with amplifications being the most abundant alteration. Out of the seven datasets, three datasets showed significant negative correlation of GSK3A alterations with overall and disease-free survival in breast cancer patients. Kaplan-Meier curve analysis demonstrated that amplification of GSK3A gene is significantly correlated with poor overall and disease-free survival of breast cancer patients, specifically in estrogen receptor positive (ER+) tumors. Further, missense and truncating mutations leads to poor disease-free survival in patients with ER+ tumors and truncating mutation showed poor overall survival in patients with Her2+ tumors. Collectively, analysis of GSK3A genomic profile in breast cancer patients provides a novel insight that GSK3A is crucial in the biological behavior and/or pathogenesis of breast cancer and hence is an important therapeutic target for breast cancer treatment.

Citation Format: Jasmeet Kaur, Karandeep Kaur. Glycogen synthase kinase 3A (GSK3A) gene alterations are negatively correlated with overall and disease-free survival in breast cancer patients: In silico analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4389.
Abstract 4396: A bioinformatics approach for cancer immunotherapeutic target identification by evaluating surfaceome gene and protein expression (CITIESGAPE) in tumor and pan-normal tissues
Authors: Tian Xiangjun , Wang Jing , Wang Yifei , Zhang Zhongting , Roth Michael , Gill Jonathan B. , Gorlick Richard
Published: September 2020

Targeted therapeutic strategies and some immunotherapies for cancer including but not limited to therapeutic antibodies, antibody-drug conjugates (ADCs) and CAR-T cells therapy demand precisely targeting cell-surface proteins that are distinctively expressed by cancer cells. We developed a robust analytical tool for cancer immunotherapeutic target identification by evaluating surfaceome gene and protein expression (CITIESGAPE) that are uniquely overrepresented by the tumor in contrast to pan-normal tissues. The first step was to run differential expression testing on RNA-seq transcriptome of cancer (for example, data from TARGET and TCGA) against each normal tissue (31 tissues from GTEx_v8), from which candidate genes were defined as those are higher expressed in tumor than all normal tissues (minimum LogFC > 1 and maximum q < 0.05). We then validated the protein expression level of candidate genes through the Human Protein Atlas (HPA) and ProteomicsDB (The latter includes mass spectrometry-based label-free proteomic data of some tumor cells and 39 normal tissues). The final and crucial step was to run surfaceome protein predicting using the COMPARTMENTS database, the HPA database and the Membranome database (v2.0), depending on an optimized confidence score. We have applied this analytical pipeline to search surfaceome proteins that are unique to Osteosarcoma, a common primary malignant bone tumor in children and young adults, and obtained 57 candidates that are potential immunotherapeutic targets. To date, eight of the top confident candidates have been further validated in clinical samples, patient-derived cell lines and xenograft models. We believe this robust analytical tool could be adaptable to other types/subtypes of cancer in searching of candidate surfaceome proteins for targeted therapy and immunotherapy, as well as precision medicine and personalized therapy.

Citation Format: Xiangjun Tian, Jing Wang, Yifei Wang, Zhongting Zhang, Michael Roth, Jonathan B. Gill, Richard Gorlick. A bioinformatics approach for cancer immunotherapeutic target identification by evaluating surfaceome gene and protein expression (CITIESGAPE) in tumor and pan-normal tissues [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4396.
Abstract 4400: Translational drug interaction database (TDID): A knowledgebase for drug interactions
Authors: Zhang Shijun , Wu Heng-Yi , Vanam Rohith , chien-WeiChiang  , Wang Lei , Li Lang
Published: September 2020

To help physicians and basic scientists in identifying drug-drug interaction mechanisms (DDI) and associated toxicity, we developed ‘The Translational Drug Interaction Database (TDID)', which is a fully searchable web-based resource that has a large collection of drug interaction data. The current TDID is seeded with pharmacokinetics (PK) or pharmacodynamics (PD) based drug interaction evidences that are extracted and classified from PubMed (from 1975 to 2015) via text mining. The DDI evidences are further classified into the following categories: PK in vitro, PD in vitro, PK in vivo, PD in vivo, clinical safety, and case report. Most importantly, cancer drug interaction clinical safety data are mined and manually curated from full-text articles. TDID also includes PK or PD DDI statements from DrugBank, and DDI-ADE associations from FAERS. Altogether, TDID integrates 88,500 pieces of DDI evidences from PubMed, 1,048,000 DDI statements from DrugBank, and over 100,000,000 DDI-ADE pairs from FAERS. Specifically, TDID has 1,480 pieces of cancer DDI toxicity evidences. These cancer DDI toxicity evidences are annotated with patient characteristics, treatment plan including dosages, and toxicity level. The database can be searched by drug name (i.e. single drug, drug pair), and the DDI query results can be sorted by study type. The Translational Drug Interaction Database (TDID) offers user-friendly browsing, searching, and filtering functions. The Translational Drug Interaction Database is available at https://tdid.bmi.osumc.edu.

Citation Format: Shijun Zhang, Heng-Yi Wu, Rohith Vanam, chien-WeiChiang, Lei Wang, Lang Li. Translational drug interaction database (TDID): A knowledgebase for drug interactions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4400.
Abstract 4410: Automatic detection of consensus gene clusters across multiple single-cell datasets
Authors: Song Qianqian , Su Jing , Miller Lance D. , Zhang Wei
Published: September 2020

Identifying co-expressed gene clusters can provide evidence for cellular activities. Thus, co-expression clustering will be a routine step in single-cell RNA-seq data analysis. We show that commonly used clustering methods for bulk-seq data are not suitable for single-cell data, and produce results that substantially disagree in the biological expectations of co-expressed genes. Herein, we present scLM, a tailored method for single-cell data, to extract co-expressed gene clusters matching the biological expectations and outperform widely used methods. Additionally, scLM can simultaneously cluster multiple single-cell datasets, i.e. consensus clustering, enabling users to leverage the large quantity of public data for novel comparative analysis. ScLM takes raw count data as input and preserves biological variations without being influenced by batch effects from multiple datasets. Results from both simulation data and real experimental data demonstrate that scLM outperforms the existing methods with considerably improved accuracy. To illustrate the biological insights of scLM, we apply it to our in-house and public experimental scRNA-Seq datasets. ScLM identifies novel functional gene modules and refines cell states, which facilitates mechanism discovery and understanding of complex biosystems such as cancers.

Citation Format: Qianqian Song, Jing Su, Lance D. Miller, Wei Zhang. Automatic detection of consensus gene clusters across multiple single-cell datasets [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4410.
Abstract 4411: A gene set enrichment analysis approach in single-cells along pseudotime trajectories reveals the dynamic activity of oncogenic pathways
Authors: Wenzel Alexander T. , Champa Devora , Howell Stephen B. , Mesirov Jill P. , Harismendy Olivier
Published: September 2020

Single cell RNA-sequencing (scRNA-seq) has advanced studies of heterogeneous tissues and cell populations in both healthy and disease states. Dimensionality reduction methods leverage this highly granular data to identify cellular states not captured by “bulk” gene expression or to organize cells along ‘pseudotime' trajectories related to biological processes such as cell cycle or cellular differentiation. Cellular states and trajectories are often annotated using individual marker genes and not gene sets or pathways, despite their more reliable and interpretable association to biological processes. Gene Set Enrichment Analysis (GSEA) was designed to test the enrichment of gene sets in bulk transcriptomic data, but due to the sparsity of the expression data, has not been adapted to single cell analysis. We propose a method to compute gene set enrichment for scRNA-seq data, which calculates an enrichment score for each cell in the dataset. We apply this method to a set of matched ovarian cancer cell lines with acquired resistance to carboplatin. After aligning the cells to a cell-cycle derived pseudotime trajectory, the activity of pathways which were dysregulated in bulk expression profiles appeared to change with the cell cycle progression. The analysis further identified interferon alpha signaling induction in resistant cells, independent of differences in cell cycle progression. Hence, by quantifying and analyzing pathway-level transcriptional activity in the context of a single-cell pseudotime trajectory, the proposed method provides more interpretable annotation of biological processes altered in cancer progression and treatment.

Citation Format: Alexander T. Wenzel, Devora Champa, Stephen B. Howell, Jill P. Mesirov, Olivier Harismendy. A gene set enrichment analysis approach in single-cells along pseudotime trajectories reveals the dynamic activity of oncogenic pathways [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4411.
Abstract 4419: Subtyping of HNSCC single-cell RNA-seq identifies transcriptional programs characterized by suppression of Mtorc1 and Wnt signaling pathways and better patient prognosis
Authors: Reed Eric , Owa Takashi , Nomoto Kenichi , Varelas Xaralabos , Kukuruzinska Maria , Monti Stefano
Published: September 2020

Single-cell RNA-seq (scRNA-seq) is an emerging platform for high-throughput profiling of individual cells in a sample and is routinely employed to investigate the transcriptional landscapes of the cellular constituents of tumors. To this end, many scRNA-seq specific clustering algorithms have emerged to analytically partition cells into modules of comparatively similar profiles. To facilitate data driven molecular subtyping of such scRNA-seq clustering results and other large-scale-omics studies, we have developed K2 Taxonomer. K2 Taxonomer is an R package built around a novel top-down hierarchical clustering algorithm, utilizing repeated perturbations of the data to generate robust taxonomical partitions of observations. The software runs additional analyses to define gene co-expression signatures of these modules, as well as to integrate user-input annotations of genes and/or observations. An interactive web portal has been generated to assist in the interrogation of the full compendium of results.

We applied K2 Taxonomer to publicly available HNSCC scRNA-seq data, identifying pertinent tumor cell subtypes, distinguished by cell cycle and epithelial-to-mesenchymal transition, and with analytical projection of this signature onto TCGA-HNSCC bulk RNA-seq data exhibiting association with worse survival in TCGA-HNSCC patients. A transcriptional signature corresponding to suppression of Mtorc1 and Wnt/β-catenin signaling was also identified in a sub-population of HNSCCs, and shown to be associated with improved patient survival. Of notice, highly ranked markers of this signature are significantly associated with gene expression changes altered by E7386 - a novel β-catenin/CBP modulator with an activity profile that closely overlaps with that of ICG-001, but exhibits ~50-100-fold lower EC50 values - suggesting a role for this signaling axis in subsets of HNSCC. In conclusion, taxonomical subtyping with K2 Taxonomer provides a novel framework to expand the scope of applicability of scRNA-seq clustering results, and has revealed potentially novel HNSCC subtypes that offer directions for future studies.

Citation Format: Eric Reed, Takashi Owa, Kenichi Nomoto, Xaralabos Varelas, Maria Kukuruzinska, Stefano Monti. Subtyping of HNSCC single-cell RNA-seq identifies transcriptional programs characterized by suppression of Mtorc1 and Wnt signaling pathways and better patient prognosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4419.
Abstract 4437: Anti-tumor immunotherapeutic strategy for uveal melanoma by combining photodynamic therapy with ROCK inhibitor
Authors: Kim Seohyun , Kim In-San
Published: September 2020

Uveal melanoma (UM) is the most frequent intraocular malignant tumor among adults. Although UM is a rare disease, the prognosis of patients with UM is poor since nearly 50% of patients develop metastatic disease, and the overall survival outcome is dismal. Here, we suggest a novel and optimal treatment regime for UM not only offering lower burden to patients with high applicability but also preventing lethal metastatic disease by empowering the immune system through the combination of local Photodynamic therapy (PDT) and ROCK inhibitor. PDT is a topical treatment modality with the minimally invasive therapeutic method and now broadly used in a treatment option for the eyes. PDT with chlorin e6 based formulated FIC elicits immunogenic cell death (ICD) of cancer cells, and hallmarks of ICD stimulate antigen-presenting cells (APCs). Furthermore, ripasudil, one of the ROCK inhibitors, is clinically approved for the treatment of glaucoma and ocular hypertension in the form of an eye drop solution. Ripasudil is repurposed as an anticancer compound based on the immuno-stimulatory effect of ROCK inhibition. A combination of FIC-PDT with ripasudil mediated stimulation of APCs leads primed cytotoxic T cell infiltration into the tumor microenvironment (TME) and eventually augment anti-tumor immunity. Moreover, the combinational strategy sensitizes PD-1/PD-L1 immune checkpoint blockade (ICB) therapy to improve survival rates over time and inhibit tumor growth of the distant tumor by generating a systemic anti-tumor immune response. Collectively, our therapeutic strategy may lead to a new treatment option for UM by suppressing the primary tumor effectively and preventing the metastatic disease.

Citation Format: Seohyun Kim, In-San Kim. Anti-tumor immunotherapeutic strategy for uveal melanoma by combining photodynamic therapy with ROCK inhibitor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4437.
Abstract 4438: A bispecific antibody targeting CD28H and PDL-1 is a novel and potent immunomodulator of T cell responses
Authors: Ramaswamy Madhu , Kim Taeil , Jones Desmond , Ghadially Hormas , Mahmoud Tamer , Garcia Andrew , Wilson Susan , Riggs Jeffrey , Schofield Darren , Carlesso GIanluca
Published: September 2020

The success of immune checkpoint inhibitors CTLA4 and PD-1 monoclonal antibody, both FDA approved cancer therapies, specifically counteracts inhibitory pathways to activate antigen-specific T cells. However, their success is limited by the fact that not all patients respond to immunotherapy and a variety of adverse events due to non specific and systemic T cell activation limits their administration.

A newly identified B7 family receptor, CD28H/TMIGD2, is constitutively expressed on T, pDCs and innate lymphoid cells including NK and ILCs. CD28H is also detected in TILs including the CD8+ tissue resident memory T cells (TRM), a T cell subset that correlates with better prognosis and response to immune checkpoint inhibitor therapy. Functionally, CD28H provides costimulatory function to T cells in the context of TCR signaling events during activation.

We engineered a bispecific antibody (Bis mAb) targeting CD28H and PDL1 aimed to augment T cell costimulation and NK activation. The CD28H-PDL1 Bis mAb potentiates T cell proliferative and cytokine responses in antigen-specific human T cell assays and induces human NK -mediated redirected killing of PDL1+ tumor cells. In T cells, the mechanism of action of the Bis mAb requires intracellular signaling domain of the CD28H which downmodulates SHP phosphorylation upon CD3 activation. Intriguingly, we found that the Bis mAb increases induction of CD8 TRM cells in vitro. In human TILs, where CD28H is mostly expressed on CD8 TRM in tumors, the Bis mAb enables higher cytokine responses over PDL1 mAb alone. Given the recent relevance of TRM cells as an important pool of anti-tumor T cell immunity, the rationale for targeting this population via CD28H in the context of blocking PDL1 may prove to be a therapeutic tool for enhancing responses to checkpoint inhibitor therapy.

Citation Format: Madhu Ramaswamy, Taeil Kim, Desmond Jones, Hormas Ghadially, Tamer Mahmoud, Andrew Garcia, Susan Wilson, Jeffrey Riggs, Darren Schofield, GIanluca Carlesso. A bispecific antibody targeting CD28H and PDL-1 is a novel and potent immunomodulator of T cell responses [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4438.
Abstract 4449: Plinabulin, a microtubule destabilizing agent, improves tumor control by enhancing dendritic cell maturation and CD8 T cell infiltration in combination with immunoradiotherapy
Authors: Neri Shinya
Published: September 2020

There are theoretical benefits of combining immune checkpoint blockade with radiotherapy, however, there are few indications that such combinations have translated to significant clinical benefit to patients. There is an unmet need to further enhance the effectiveness of these “immunoradiotherapy” combinations. We hypothesize that enhancing antigen presentation through augmenting dendritic cell (DC) activation could potentially improve the effectiveness of immunoradiotherapy. Towards this end, we have investigated the potential for combining the microtubule destabilizing first-in-class agent Plinabulin as the candidate DC activator, with radiotherapy and anti-PD1 immunotherapy (αPD1). First, we investigated how DCs could be affected by Plinabulin and irradiation in in vitro assays. We irradiated SP37A3 and XS106 DC lines with 10Gy and added Plinabulin to those cells. With the addition of Plinabulin, the phosphorylation of JNK and NFκB was upregulated and DC maturation was promoted with high MHC-II, CD86, CD80 and CD40 surface expressions (p<0.05, Dunnett's test). Combining Plinabulin and radiotherapy further augmented the response, particularly in sequence with plinabulin but not in combination. In in vivo models. TSA syngeneic breast cancer cells were implanted to the flank of BALB/c mice, irradiated with 24Gy/3fr, and treated with αPD1 and Plinabulin. There were no anti-tumoral effects with αPD1 alone, Plinabulin alone, and αPD1+Plinabulin groups. However, triple therapy showed much stronger tumor regression than the double therapy of irradiation and αPD1. Tumors were collected from the mice at 2.5-3 weeks after the irradiation and dissociated tumor cells were analyzed for DC activation and immune repertoire using flow cytometry. The percentage of CD8 T cells and CD86+ DCs in the tumor were significantly increased in the triple therapy group that were not seen for the monotherapy or bimodal therapy groups (p<0.05, Dunnett's test). Our findings may serve as a rationale for establishing clinical trial designs of synergistic combinational approaches to enhance immunoradiotherapy.

Citation Format: Shinya Neri. Plinabulin, a microtubule destabilizing agent, improves tumor control by enhancing dendritic cell maturation and CD8 T cell infiltration in combination with immunoradiotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4449.
Abstract 4458: New HDAC inhibitor (M166) has a synergistic effect with immune checkpoint inhibitor in lung cancer treatment
Authors: Lee Sung Yong , Shin Hyun-Seock , Choi Juwhan
Published: September 2020

Recently, Immune checkpoint inhibitor (ICI) has been widely applied to various types of cancer therapies and it is used as a treatment in clinical application. ICI has a higher effect on lung cancer than other cancers; however, the treatment efficiency is only about 25%. Therefore, many research groups are making efforts to improve treatment efficiency with co-treatment therapy using chemical medications and ICIs. We evaluated the efficacy of a new PAN-HDAC inhibitor (M166) and ICI. M166 showed growth inhibition in mouse lung cancer cell (TC-1) line, mouse lung carcinoma (LLC) and human lung cancer cell (A549) line compared with normal epithelial cell lines (NIH3T3 and HEK293. Also, we checked ten kinds of immune-related cytokines and chemokines in mRNA level by treated with M166 using RT-PCR methods. CCL2 and CXCL10 chemokines are significantly increased by dose-dependently. These results indicate that M166 regulates immune response by CCL2 and CXCL10 chemokine related pathways. In order to check the synergistic effect of ICI plus HDACi co-treatment, we explored the employment of M166 in combination with ICI in a preclinical mouse model. For in vivo experiment, the mice were divided into four groups: group 1 received no treatment after the E7-expressing TC-1 tumor challenge, group 2 was treated with M166, group 3 was treated with mouse PD-1 antibody, and group 4 was both treated with M166 and mouse PD-1 antibody. The combination therapy of M166 and mouse PD-1 antibody significantly inhibited tumor growth. These results indicate that ICI plus HDACi treatment induces tumor growth inhibition through an immune response. These findings have important implications for future clinical translation.

Citation Format: Sung Yong Lee, Hyun-Seock Shin, Juwhan Choi. New HDAC inhibitor (M166) has a synergistic effect with immune checkpoint inhibitor in lung cancer treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4458.
Abstract 4479: Immune exhaustion of tumor microenvironment mediates crizotinib resistance in ALK rearranged advanced non small cell lung cancer
Authors: Zhang Yongchang
Published: September 2020

Introduction: Previous study showed the correlation between immune tumor microenvironment and the efficacy of checkpoint inhibitor in non-small cell lung cancer (NSCLC). This study evaluated whether biomarkers for immune tumor microenvironment including programmed death ligand 1 (PD-L1), cluster of differentiation 8 (CD8), Granzyme B (GZMB) and T cell receptor (TCR) could predict the treatment response to the first-line crizotinib of patients with ALK-rearranged non-small cell lung cancer (NSCLC).

Methods: We retrospectively evaluated patients with advanced NSCLC who received crizotinib at Hunan Cancer Hospital between January 2016 and December 2018. The patients' baseline expression of PD-L1, CD8 and GZMB was evaluated in one slide using immunofluorescence. TCR was also evaluated with paired blood samples simultaneously.

Results: All the 108 eligible patients were stratified according to their expression profile into PD-L1+/CD8+/GZMB+ (n=21), PD-L1-/CD8+/GZMB+ (n=42), PD-L1+/CD8-/GZMB- (n=23), and PD-L1-/CD8-/GZMB- (n=22) group. Patients with strong PD-L1 expression have a significantly inferior objective response rate (36.5% versus 72.2%, p = 0.002), and shorter progression-free survival (PFS) (5.6 versus 16.5 months, p < 0.001) as compared to patients with weak or negative PD-L1 expression. Among the four groups, we found patients with PD-L1-/CD8+/GZMB+ had the best response (85.7% versus 26%, p=?), while PD-L1+/CD8-/GZMB- showed primary resistance to crizotinib had the worse outcomes (4.6 versus 18 months, p < 0.001). Furthermore, for TCR analysis, the counts of TCR and Shannon index was strongly correlated with longer PFS for crizotinib treatment (20.6 versus 8.2months, p < 0.001; 19.3 versus 6.1 months, p < 0.001, respectively). However, the clonality acts as poor predictive marker with worse PFS (6.8 versus 18.2 months, p < 0.001). Finally, three patients with de novo resistance to crizotinib experienced a favorable response to anti-PD-1 therapy.

Conclusions: This study revealed immune exhaustion for tumor microenvironment as a poor predictive marker in first-line crizotinib-treated ALK-rearranged non-small cell lung cancer. The findings indicate the reshaping of an inflamed immune phenotype characterized by PD-L1, CD8, GZMB, and TCR expression and suggest potential therapeutic sensitivity to PD-1 blockade.

Citation Format: Yongchang Zhang. Immune exhaustion of tumor microenvironment mediates crizotinib resistance in ALK rearranged advanced non small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4479.
Abstract 4484: ScRNA-seq of patients with immune-related adverse events and checkpoint inhibitor therapy
Authors: Khan Shaheen , Gerber David E.
Published: September 2020

Despite the remarkable success of immune checkpoint inhibitor (ICI) therapy, a significant number of patients develop severe and unpredictable immune-related adverse events (irAEs) affecting a wide variety of organs. Concerns over irAE have led to the exclusion of patients with autoimmune disease from ICI clinical trials. Role of host genetic and immune factors in mediating irAEs remain unclear and it is not clear if the manifestations of irAEs is associated with response to therapy. In this study, we used single cell RNA sequencing (ScRNA-seq) to study the peripheral immune repertoire at a single cell level to identify blood-based signatures predictive of irAEs and response to ICI therapy. We characterized changes in host immune system in 40 patients receiving ICI therapy by performing ScRNA-seq on peripheral blood mononuclear cells (PBMCs) at baseline and post-immunotherapy. In addition, we evaluated serum levels of 40 cytokines/chemokines, antinuclear autoantibodies (ANA) and 124 autoantibodies and performed bulk RNA sequencing on PBMCs at baseline and post immunotherapy in these patients. Our preliminary data analysis identified signatures of autoantibodies and cytokines correlating with response and toxicity. ScRNA-seq data revealed changes in distinct immune cell subsets and gene expression signatures in patient developing irAEs. In this meeting, we will present preliminary ScRNA-seq data combined with immune and genetic correlates of irAEs and response to therapy in our patient cohort. We hope that our studies can help identify blood-based biomarker signatures predictive of irAEs and/or response and reveal novel insights into the mechanisms underlying irAE. Our current genetic data suggests further expanding the genetic studies in larger patient population to identify the role of underlying genetic predisposition to autoimmunity in mediating irAEs. We hope our findings may ultimately help customize therapy, expand use of immunotherapy and prevent toxicities.

Citation Format: Shaheen Khan, David E. Gerber. ScRNA-seq of patients with immune-related adverse events and checkpoint inhibitor therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4484.