bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 107: Reciprocal Sox2 regulation by SMAD1 and SMAD3 is critical for anoikis resistance and metastasis in ovarian cancer
Authors: Shonibare Zainab M. , Monavarian Mehri , O'Connell Kathleen , Altamore Diego , Shelton Abigail , Mehta Shubham , Jaskula-Sztul Renata , Phaeton Rebecca , Berchuck Andrew , Nixon Andrew B. , Arend Rebecca , Lee Nam Y. , Miller Ryan , Hempel Nadine , Mythreye Karthikeyan
Published: June 2022

The tumor ascites environment is enriched with cytokines and growth factors especially members of the Transforming Growth Factor-β (TGF-β) family, that play crucial roles in modulating anoikis sensitivity and consequently metastatic progression in ovarian cancer. Here, we demonstrated for the first-time significant broad dichotomy between these TGF-β members: BMPs and TGF-β/activin and their regulated SMADs in this process. We previously reported BMP9/GDF2 to be in low to undetected levels in ovarian cancer patient-derived ascites and promoted anoikis resistance in both breast and ovarian cancer cell lines. We have now identified additional BMPs -BMP2, BMP4 and BMP9 as promoting anoikis sensitivity in a spectrum of ovarian cancer cells. Conversely, we found TGF-βs (1 and 2) to be in much higher expression and demonstrated that TGF-β and activin promoted anoikis resistance in ovarian cancer. Hence, in an attempt to identify genes downstream of BMPs that may provide anoikis resistance, transcriptomics was performed leading to the identification of Sox2, a developmental gene with prior established roles in OVCA, as being significantly downregulated in response to BMP9. We further uncovered Sox2 to be reciprocally regulated by anoikis-promoting BMPs (2, 4 and 9) and anoikis suppressing TGF-β and activin A. Our findings highlight a novel contrasting SMAD dependent regulation of Sox2 as a central node for controlling tumor cell survival in ovarian cancers demonstrating a subset of BMPs as a therapeutic strategy in cancer. The studies presented here begin to elucidate the mechanisms of reciprocal epigenetic regulation of Sox2 by BMP and TGF-β/activin in the context of ovarian cancer, which is the most lethal gynecologic malignancy in women due to peritoneal metastatic spread.

Citation Format: Zainab M. Shonibare, Mehri Monavarian, Kathleen O'Connell, Diego Altamore, Abigail Shelton, Shubham Mehta, Renata Jaskula-Sztul, Rebecca Phaeton, Andrew Berchuck, Andrew B. Nixon, Rebecca Arend, Nam Y. Lee, Ryan Miller, Nadine Hempel, Karthikeyan Mythreye. Reciprocal Sox2 regulation by SMAD1 and SMAD3 is critical for anoikis resistance and metastasis in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 107.
Abstract 1081: Design of combinatory RNA molecules to simultaneously activate tumor immunity and target several signaling pathways in solid tumors
Authors: Hillmann Petra , Selvaraj Justin A. , Zimmerli Sina E. , Birrer Pascale G. , Bohnert Claudia C. , Zuideveld Klaas P. , Scherer Stefan J. , Metzger Friedrich
Published: June 2022

Combination therapy is implicated for many oncological indications to circumvent resistance mechanisms that reduce efficacy of targeted drugs. In recent years, mRNA has been successfully utilized preclinically to deliver cytokines to tumors locally at efficacious doses.

Versameb has developed a technology platform which enables combinatorial approaches for intra-tumoral application of nucleotide therapeutics that comprise an mRNA element covalently linked to up to three independent siRNA elements targeting various tumor-relevant genes. Such constructs allow a dose-synchronized treatment of different components in parallel, which opens the opportunity to address up to four cancer targets within a single RNA molecule.

For proof of concept, we designed a combinatory molecule consisting of mRNA coding for human interleukin 2 (IL-2) linked to a small interfering RNA (siRNA) targeting vascular endothelial growth factor A (VEGFA). Transfection of A549 adenocarcinoma cells led to dose-dependent secretion of efficacious concentrations of IL-2 and simultaneous complete VEGFA knockdown. IL-2 protein functionality was tested in SK-OV-3 cell spheroids co-cultured with human primary peripheral blood mononuclear cells (PBMCs). After treatment, secreted IL-2 protein induced immune cell activation and consecutive spheroid killing by PBMCs from three independent donors. VEGFA functionality was tested in an in vitro vascularization assay in HUVEC cells. Cells were treated with supernatants of vehicle or RNA transfected SCC-4 head & neck cancer cells producing high endogenous VEGFA, and vascular sprouting was analyzed quantitatively. While vehicle-treated cell supernatant induced vessel sprouting, supernatant from RNA treated cells showed much reduced sprouting in vitro consistent with the lower VEGFA levels measured, confirming functionality of the siRNA effect on VEGFA protein expression.

We further provide examples of combinatory molecules targeting up to 4 independent key cancer hallmarks and their functional outcomes in various cancer cell types.

In conclusion, mRNA combination constructs can be developed into cancer therapeutics that target several pro-tumorigenic pathways simultaneously with a single molecule overcoming hurdles of delivery, toxicity, and regulatory requirements for combination therapies.

Citation Format: Petra Hillmann, Justin A. Selvaraj, Sina E. Zimmerli, Pascale G. Birrer, Claudia C. Bohnert, Klaas P. Zuideveld, Stefan J. Scherer, Friedrich Metzger. Design of combinatory RNA molecules to simultaneously activate tumor immunity and target several signaling pathways in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1081.
Abstract 1087: Development and characterization of a bispecific antibody (GB265) against PD-L1 and TIGIT
Authors: Huang Su , Yang Riyao , Huang Cai , Putrevu Saroja , Fay Nathan S. , Wright Kimberly , Zaman Mohd Saif , Huang Betty , Wang Bo , Wright Meredith , Wang Yanan , Tian Xuefei , Liu Yue
Published: June 2022

Background: Cancer immunotherapy has transformed cancer therapy. In particular, blockade of the PD-L1/PD-1 axis with PD-1 or PD-L1 antibodies has achieved successful and durable treatment outcomes in some patients across a variety of cancer types. However, primary or acquired resistance to immunotherapy and adverse effects have been observed in cancer patients, due to a hostile TME and the immunosuppressive activity of various inhibitory checkpoint receptors and their ligands on immune cells and cancer cells. It was recently reported that dual blockade of PD-1/PD-L1 and TIGIT/CD155 could improve treatment efficacy in NSCLC patients. We therefore develop bispecific antibody against both PD-L1 and TIGIT with the aim of enhancing the efficacy of therapy and mitigating potential adverse effects.

Methods: We obtained TIGIT binders by VHH library panning, and constructed PD-L1/TIGIT BsAbs of two different structures either by fusing the TIGIT VHH to a PD-L1 IgG or by fusing the TIGIT VHH to a PD-L1 VHH-Fc. TIGIT binding and CD155 blocking was analyzed by fluorescence-activated cell sorting (FACS) using Jurkat cells stably expressing TIGIT. Function assays were conducted with a co-culture system of H1975 stably expressing OKT3 and induced human exhausted T cells or luciferase reporter Jurkat cells with high expression of PD-1 and TIGIT.

Results: Both bispecific antibodies effectively bind TIGIT on T cells and PD-L1 on PD-L1 positive cancer cells. Interaction between BsAb and the two targets bridge the T cell and the cancer cell and result in the clustering of the antibody-antigen complexes at the contact area of the two cells. Functionally, GB265 antibodies effectively blocked CD155 and PD-1 signal pathways in the cell-based reporter system and exhibited ADCC function specifically to target cancer cells.

Conclusion: GB265 antibodies have the potential to outperform PD-L1 and TIGIT monospecific antibodies or their combinations in rejuvenating anti-tumor immunity in cancer patients due to its bridging activity and dual blockade function.

Citation Format: Su Huang, Riyao Yang, Cai Huang, Saroja Putrevu, Nathan S. Fay, Kimberly Wright, Mohd Saif Zaman, Betty Huang, Bo Wang, Meredith Wright, Yanan Wang, Xuefei Tian, Yue Liu. Development and characterization of a bispecific antibody (GB265) against PD-L1 and TIGIT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1087.
Abstract 1094: SMARCA4-mediated chromatin remodeling regulates osimertinib resistance in EGFR-mutant lung adenocarcinoma
Authors: de Miguel Fernando J. , Cai Wesley L. , Melnick Mary Ann , Robles-Oteiza Camila , Wurtz Anna , Toki Maria I. , Rimm David L. , Homer Robert , Nguyen Don X. , Politi Katerina A.
Published: June 2022

Targeted therapies have transformed the clinical management of many types of tumors, including EGFR-mutant lung adenocarcinomas. Although these treatments are highly beneficial for patient survival, acquired resistance almost inevitably occurs limiting cures with these agents. The tyrosine-kinase inhibitor (TKI) osimertinib is approved for the first-line treatment of EGFR-mutant lung tumors and leads to increased progression-free and overall survival compared to earlier generations of TKIs. However, acquired resistance to osimertinib frequently occurs and is mostly due to off-target mechanisms, many of which are not well-understood. Therefore, it is critical to understand the molecular processes behind these mechanisms of resistance.

We generated osimertinib resistant cell lines and patient-derived models to identify and study novel mechanisms of osimertinib resistance. We found that, in some osimertinib resistant tumors, SMARCA4 is stabilized upon osimertinib treatment and knock-down of SMARCA4 re-sensitizes the resistant cells to osimertinib and globally alters their chromatin profile. ATAC-Seq and RNA-Seq studies revealed that SMARCA4 alters chromatin accessibility in resistant tumors to maintain the transcriptional activity of genes involved in cell proliferation. Furthermore, SMARCA4 enables access to NRF2 binding sites enhancing an antioxidant response necessary for cells to tolerate the increase in reactive oxygen species and oxidative stress created by osimertinib. These processes converge and lead to an increase in the amount of DNA damage that is repaired by ATR. Indeed, we found that these osimertinib resistant tumors are vulnerable to ATR inhibition.

In summary, we have identified a new epigenetic mechanism of resistance to osimertinib driven by SMARCA4 that generates a vulnerability to ATR inhibition, offering new approaches to target TKI-resistant tumors.

Citation Format: Fernando J. de Miguel, Wesley L. Cai, Mary Ann Melnick, Camila Robles-Oteiza, Anna Wurtz, Maria I. Toki, David L. Rimm, Robert Homer, Don X. Nguyen, Katerina A. Politi. SMARCA4-mediated chromatin remodeling regulates osimertinib resistance in EGFR-mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1094.
Abstract 1123: c-Met downregulation contributes to Metformin-induced inhibition of MDA-MB-468 triple negative breast cancer cells
Authors: Gant Dana M. , Ma Zhikun , Parris Amanda B. , Yang Xiaohe
Published: June 2022

Triple negative breast cancer (TNBC) is the breast cancer subtype that is most refractory to treatment. Thus, identification of novel molecular targets and drug treatments are needed to improve prognosis for patients. The widely used anti-diabetic drug Metformin has been reported to possess anti-cancer properties. Although it is believed that Metformin may exert its anti-cancer effects through indirect reduction of glucose and insulin levels, and directly activating the AMPK kinase and inhibiting downstream cancer-related pathways, the molecular mechanisms underlying its anti-cancer function in TNBC have yet to be fully elucidated. In this study, we investigated the role and mechanism of Metformin-mediated downregulation of oncogenic c-Met in MDA-MB-468 TNBC cells. Our results demonstrated greater sensitivity of MDA-MB-468 cells to Metformin compared to other breast cancer cell lines. Metformin-induced inhibition of MDA-MB-468 cell proliferation was correlated with the downregulation of c-Met at both protein and mRNA levels. Overexpression of c-Met in MDA-MB-468 cells using a lentiviral vector system rendered the cells resistant to Metformin, which was accompanied by the promotion of cancer stem cell stemness. Functionally, we demonstrated that c-Met induced activation of receptor tyrosine kinase (RTK) signaling pathways play a critical role in c-Met-modulated Metformin responsiveness. These novel findings highlight c-Met as a key regulator of Metformin-mediated inhibition of TNBC cell growth, suggesting that c-Met overexpression may be a critical factor contributing to metformin resistance. To this end, combination of Metformin with c-Met inhibitors could be a useful strategy to improve Metformin-mediated anti-cancer efficacies in TNBC.

Citation Format: Dana M. Gant, Zhikun Ma, Amanda B. Parris, Xiaohe Yang. c-Met downregulation contributes to Metformin-induced inhibition of MDA-MB-468 triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1123.
Abstract 1149: Decitabine inhibits breast cancer cell proliferation through inhibition of endogenous retroviruses in vitro
Authors: Li Jiayi , Lin John , Andrada Leo , Grohol Bryce , Nong Serratt , Liu Yingguang
Published: June 2022

The purpose of the study is to elucidate the antineoplastic mechanisms of decitabine, a DNA methyltransferase inhibitor. Previous literature suggests that decitabine activates human endogenous retroviruses which subsequently enhances expression of interferons, thereby suppressing tumor cell proliferation. Contrary to this hypothesis, our in vitro study with human and mouse breast cancer cell lines showed a positive correlation between the antiproliferative effect of decitabine and its suppressive effect on two endogenous retroviruses, the human endogenous retrovirus type K (HERV-K) and the mouse mammary tumor virus (MMTV). Decitabine stimulated proliferation of the T47D human breast cancer cell line at lower doses but inhibited cell proliferation at higher doses. Correspondingly, HERV-K transcripts (env and pol) increased at lower doses and decreased at higher doses. Interestingly, expression of the E-cadherin gene, an indicator of epithelial differentiation, followed an opposite trend. Decitabine inhibited proliferation of the mouse 4T1 breast cancer cell line and its expression of MMTV in a dose-dependent manner, while enhancing expression of the mouse E-cadherin. 4T1 cells overexpressing MMTV env became more resistant to decitabine, so was a human breast cancer cell line (BT-20) overexpressing HERV-K env, while T47D cells with HERV-K knockdown became more susceptible to the drug. Meanwhile, in vivo studies showed that decitabine successfully suppressed tumor development in BALB/c mice inoculated with 4T1 cells. Surprisingly, expression of MMTV env was elevated in tumors of mice treated with decitabine, along with enhanced E-cadherin expression. These data suggest decitabine inhibits cancer cell proliferation (at least partially) through inhibition of endogenous retroviruses in vitro, but its mechanisms of action in vivo are different. Further studies of the interaction between decitabine and endogenous retroviruses using engineered 4T1 cells in BALB/c mice are currently underway.

Citation Format: Jiayi Li, John Lin, Leo Andrada, Bryce Grohol, Serratt Nong, Yingguang Liu. Decitabine inhibits breast cancer cell proliferation through inhibition of endogenous retroviruses in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1149.
Abstract 1151: Pharmacogenomics genotyping from clinical somatic whole exome sequencing: Aldy, a computational tool
Authors: Osei Wilberforce A. , Shugg Tyler , Ly Reynold C. , Bray Steven M. , Salisbury Benjamin A. , Ratcliff Ryan R. , Pratt Victoria M. , Numanagić Ibrahim , Skaar Todd
Published: June 2022

Background Pharmacogenomics (PGx) testing can reduce toxicities and improve efficacy of several drugs used to treat cancer and associated symptoms. PGx results can be determined from germline whole-exome sequencing (WES), but somatic mutations may cause discordance between tumor and germline DNA. Since clinical diagnostic sequencing in oncology frequently only includes tumor DNA, there would be clinical value in calling germline PGx genotypes from tumor DNA. Thus, the purpose of this study was to assess the feasibility of using somatic WES data to call germline PGx genotypes.

Methods Germline and somatic WES data were obtained as part of the clinical workflow for 64 patients treated at the solid molecular tumor board clinic at Indiana University. Aldy v3.3 was implemented in LifeOmic’s Precision Health Cloud™ to call PGx genotypes from somatic WES. Somatic Aldy calls were compared with previously validated Aldy germline calls for 8 genes: CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, CYP4F2, DPYD, and TPMT. Somatic read depth was >100x, except for the intronic CYP3A4*22 variant, which was >30x.

Results Somatic and germline Aldy calls were compared for a total of 512 genotypes and 56 (11%) calls were discordant. Discordant calls were most common for CYP2B6 (23.4%), followed by CYP2D6 (14.1%), CYP2C19 (10.9%), CYP2C8 (6.3%), and DPYD (6.3%). In contrast, all Aldy calls were concordant for CYP3A5 and TPMT. 38 out of 64 subjects (59%) had discordant calls for at least one gene. The most common first cancer diagnoses in our cohort were colorectal (9.3%), breast (7.8%), and pancreatic (7.8%), and the rates of discordant Aldy calls did not differ by cancer type (p>0.05 for all cancer types). Based on our analyses of discordant calls, we anticipate that adjusting Aldy’s thresholds for variant calling may allow Aldy to determine genotypes from somatic WES data.

Conclusion In most cases, genotype calls of drug metabolism genes from tumor DNA reflected the germline genotypes; however, additional work needs to be done to determine if the remaining discordant calls can be corrected by modifying the informatics tools or if they are due to somatic mutations.

Citation Format: Wilberforce A. Osei, Tyler Shugg, Reynold C. Ly, Steven M. Bray, Benjamin A. Salisbury, Ryan R. Ratcliff, Victoria M. Pratt, Ibrahim Numanagić, Todd Skaar. Pharmacogenomics genotyping from clinical somatic whole exome sequencing: Aldy, a computational tool [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1151.
Abstract 1156: Genome Nexus: A comprehensive resource for the annotation and interpretation of genomic variants in cancer
Authors: de Bruijn Ino , Li Xiang , Sumer Onur , Gross Benjamin , Sheridan Robert , Ochoa Angelica , Wilson Manda , Wang Avery , Zhang Hongxin , Lisman Aaron , Abeshouse Adam , Rodenburg Sander , van Hagen Sjoerd , Fijneman Remond , Meijer Gerrit , Schultz Nikolaus , Gao Jianjiong
Published: June 2022

Interpreting genomic variants in tumor samples presents a challenge in research and the clinical setting. A major barrier is that information about variants is fragmented across disparate databases, and aggregating information from these requires building extensive infrastructure. To this end, we have developed Genome Nexus, a one stop shop for variant annotation, equipped with a powerful API for bulk annotation of variants and a user friendly interface for cancer researchers.

Genome Nexus is available at https://www.genomenexus.org. It a) aggregates variant information from a large number of sources that are relevant to cancer research and clinical applications; b) allows high-performance programmatic access to the aggregated data via a unified API; c) provides a search interface and a reference page for individual cancer variants; d) provides user-friendly tools for annotating variants in patients; e) is freely available under an open source license and can be installed in a private cloud or local environment.

Genome Nexus contains annotations from more than a dozen resources, including those that provide variant effect information (VEP), protein sequence annotation (Uniprot, Pfam, dbPTM), functional consequence prediction (Polyphen-2, Mutation Assessor, SIFT), population prevalence (gnomAD, dbSNP, ExAC), cancer population prevalence (Cancer Hotspots, SignalDB) and clinical actionability (OncoKB, CIViC, Clinvar). The annotations can be accessed through the website, the API, and a command line client.

Genome Nexus is unique in providing a user friendly interface specific to cancer that allows high performance annotation of any variant. It is the main annotation service for the popular cancer genomics tool cBioPortal, which serves thousands of users daily. It is also offered as a standalone tool for annotation, allowing researchers and clinicians as well as genomic infrastructure developers to leverage it directly in their own workflows. For example, a local installation of Genome Nexus is used for annotating all variants in AACR Project GENIE.

Citation Format: Ino de Bruijn, Xiang Li, Onur Sumer, Benjamin Gross, Robert Sheridan, Angelica Ochoa, Manda Wilson, Avery Wang, Hongxin Zhang, Aaron Lisman, Adam Abeshouse, Sander Rodenburg, Sjoerd van Hagen, Remond Fijneman, Gerrit Meijer, Nikolaus Schultz, Jianjiong Gao. Genome Nexus: A comprehensive resource for the annotation and interpretation of genomic variants in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1156.
Abstract 120: HIF-1α regulation by Smurf2-mediated protein degradation in a VHL-independent manner
Authors: Zhao Shuai , El-Deiry Wafik S.
Published: June 2022

HIF-1α is the main mediator of hypoxic response in cells and contributes to angiogenesis, metastasis and therapy resistance in cancer. The canonical pathway of HIF-1α degradation in normoxia is through the Von Hippel-Lindau (VHL) E3 ubiquitin ligase. We previously found that knockdown of CDK4 decreased HIF-1α expression in a VHL-independent manner. To explore the mechanism of CDK4-induced HIF-1α stabilization, we performed a proteomic analysis on HIF-1α-interacting proteins with palbociclib treatment, a bioinformatic prediction of HIF-1α-targeting E3 ubiquitin ligases and identified SMAD specific E3 ubiquitin protein ligase 2 (Smurf2) to be involved in a non-canonical HIF-1α regulation mechanism. Overexpression of Smurf2 enhanced the ubiquitination of HIF-1α and reduced expression of HIF-1α in HCT116 colorectal cancer cells. Smurf2 overexpression downregulated HIF-1α target gene SLC2A1 but not HIF1A mRNA level in hypoxia. Knockdown of SMURF2 increased HIF-1α expression in normoxia even when CDK4 was knocked-down. TCGA analysis showed correlation between high SMURF2 mRNA and better prognosis in kidney renal clear cell carcinoma (KIRC). In KIRC, VHL loss is a frequent mutational event that leads to high constitutive expression of HIF-1α and HIF-2α. Thus, we investigated a role for Smurf2 in HIF regulation in this cancer type. Overexpression of Smurf2 suppressed HIF-1α expression in RCC4 VHL-deficient clear cell renal cancer line in normoxia. Knockdown of SMURF2 increased HIF-1α expression in RCC4 cells. Treatment of RCC4 cells with Smurf2 inhibitor Heclin also increased HIF-1α expression in normoxia. HIF-2α expression was reduced upon Smurf2 overexpression in RCC4 cells under normoxia and in SW480 colorectal cancer cells under hypoxia. In summary, Smurf2 mediates VHL-independent HIF-1α degradation, which provides new insights for HIF-1α targeting in cancers especially with VHL deficiency, and targeting of Smurf2 to increase HIF activity may have functional consequences in non-neoplastic disorders.

Citation Format: Shuai Zhao, Wafik S. El-Deiry. HIF-1α regulation by Smurf2-mediated protein degradation in a VHL-independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 120.
Abstract 1207: Bento: A toolkit for subcellular spatial analysis of RNA organization
Authors: Mah Clarence , Ahmed Noorsher , Lam Dylan , Carter Hannah , Yeo Gene
Published: June 2022

We present Bento, a Python toolkit for subcellular spatial analysis of RNA, implementing tools for ingesting highly-multiplexed spatial transcriptomics data, visualization, detection of RNA localization patterns, identification of cell states, and subcellular spatial statistics. We applied our methods to analyze multiple spatial transcriptomics datasets from a variety of platforms, including seqFISH+ (10k RNA in cultured mouse fibroblast cells) MERFISH (130 RNA in cultured U2-OS cells), and the Spatial Molecular Imager (980 RNA in FFPE of NSCLC and breast cancer tumors) to show how subcellular spatial analysis can be leveraged to understand RNA localization and behavior. We use tensor factorization to perform unsupervised clustering analysis of subcellular patterns in each dataset, demonstrating how cell type and morphology are consistently linked to spatial RNA organization.

Citation Format: Clarence Mah, Noorsher Ahmed, Dylan Lam, Hannah Carter, Gene Yeo. Bento: A toolkit for subcellular spatial analysis of RNA organization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1207.
Abstract 1208: Denoising spatial expression profiling data based on in situ position and image information
Authors: Wang Yunguan , Song Bing , Wang Shidan , Chen Mengyi , Xie Yang , Xiao Guanghua , Wang Li , Wang Tao
Published: June 2022

Spatial expression profiling (SEP) technologies provide gene expression close to or even superior to single-cell resolution while retaining the physical locations of sequencing and often also providing matched pathology images. However, the expression data captured by SEP technologies suffer from high noise levels, including but not limited to dropouts as in regular single-cell RNA-sequencing (scRNA-seq). The extra experimental steps for preserving the spatial locations of sequencing could result in even more severe noises, compared to regular scRNA-seq. Fortunately, such noises could be largely reduced by leveraging information from the physical locations of sequencing and the tissue and cellular organization reflected by corresponding pathology images. In this work, we demonstrated the extensive levels of noise in SEP data. We developed a mathematical model, named Sprod, to reduce such noises based on latent space and graph learning of matched location and imaging data. We comprehensively validated Sprod and demonstrated its advantages over prior methods for removing dropouts in scRNA-seq data. We further showed that, after adequately de-noising by Sprod, differential expression analyses, pseudotime analyses, and cell-to-cell interaction inferences yield significantly more informative results in various biological application settings. In particular, with Sprod, we discovered 3-4 times more RNA transcripts that were actively transported in mouse hippocampus neurons. We also showed that the tumor cells at the tumor-stroma boundaries demonstrate differential transcriptomic features from the tumor cells in the central regions, caused by their interactions with the stroma/immune cells. Overall, we envision denoising by Sprod to become a key first step to empower SEP technologies for biomedical discoveries and innovations.

Citation Format: Yunguan Wang, Bing Song, Shidan Wang, Mengyi Chen, Yang Xie, Guanghua Xiao, Li Wang, Tao Wang. Denoising spatial expression profiling data based on in situ position and image information [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1208.
Abstract 1234: PD-L1 PET: A potential biomarker of checkpoint inhibitor sensitivity in renal cancer
Authors: Mulgaonkar Aditi , Woolford Layton , Elias Roy , Nham Kien , Guan Bing , Hao Guiyang , Stevens Christina , Tcheuyap Vanina Toffessi , Haldeman Sydney , Miyata Jeffrey , Carrillo Deyssy , Qin Qian (Janie) , Singla Nirmish , Bowman Isaac , Cadeddu Jeffrey , Margulis Vitaly , Christie Alana , Kapur Payal , Pedrosa Ivan , Dakanali Marianna , Oz Orhan , Sun Xiankai , Brugarolas James
Published: June 2022

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy. Program death-ligand 1 (PD-L1) and its corresponding receptor (PD-1) are the target of most FDA-approved ICIs. Perhaps unsurprisingly, PD-L1 levels are predictive of response to targeting drugs in lung and other cancers. However, PD-L1 is not predictive in other tumor types including renal cell carcinoma (RCC). PD-L1 is typically measured on tumor biopsies or archival tissues and we hypothesized that the lack of PD-L1 predictive power is due to tumor heterogeneity and tumor evolution. To test this notion, we embarked upon the generation of a PD-L1 probe that would enable real time assessment of PD-L1 across sites of disease using molecular imaging. We leveraged atezolizumab, a highly specific PD-L1 therapeutic antibody with a mutant Fc. Atezolizumab (ATZ) was labeled with 89Zr after conjugation with a bifunctional chelator and evaluated by positron emission tomography (PET). In extensive preclinical studies using multiple patient-derived xenografts (PDXs) with variable PD-L1 levels, we show that 89Zr-ATZ PET is able to distinguish RCC with variable PD-L1 expression. These data set the foundation for an investigator new drug (IND) approval from the FDA and an ongoing clinical trial in RCC patients (NCT04006522). Consistent with our hypothesis, 89Zr-ATZ PET shows substantial PD-L1 heterogeneity not only across but also within patients at different sites of disease. Furthermore, preliminary data suggest that PD-L1 levels may be predictive of response to ICI. By assessing PD-L1 expression in real time, 89Zr-ATZ PET may enable: (1) the identification of patients most likely to respond to PD-L1/PD-1 targeted therapies; (2) tailored management of the disease across different sites; (3) the evaluation of interventions that modulate PD-L1 levels; (4) insights into toxicities; and (5) probing resistance.

Citation Format: Aditi Mulgaonkar, Layton Woolford, Roy Elias, Kien Nham, Bing Guan, Guiyang Hao, Christina Stevens, Vanina Toffessi Tcheuyap, Sydney Haldeman, Jeffrey Miyata, Deyssy Carrillo, Qian (Janie) Qin, Nirmish Singla, Isaac Bowman, Jeffrey Cadeddu, Vitaly Margulis, Alana Christie, Payal Kapur, Ivan Pedrosa, Marianna Dakanali, Orhan Oz, Xiankai Sun, James Brugarolas. PD-L1 PET: A potential biomarker of checkpoint inhibitor sensitivity in renal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1234.
Abstract 1266: Circulating immune biomarkers in peripheral blood reflect the effect of a CD200 immune checkpoint targeted immunotherapy in pet dogs with high-grade glioma
Authors: Puerta Martinez Francisco W. , Schnell Sathya , Arnold Susan , Pluhar Elizabeth G. , Olin Michael
Published: June 2022

The extent to which immune cell phenotypes in the peripheral blood reflect intratumoral immune activity prior to and early in cancer immunotherapy is unclear. Here we studied the population dynamics of immune cells and immune phenotypic changes, using clinical blood samples and a multicolor flow cytometry analysis. These samples were serially obtained from a cohort of high-grade glioma canine patients enrolled in a trial with an immunotherapy that targets the CD200/CD200R immunological synapse. This therapy consists of an alternative checkpoint inhibitor peptide (CD200AR-L) that modulates the CD200 immune checkpoint, currently being used in an adult phase I safety trial for recurrent glioblastoma trial (NCT04642937) and a canine high-grade spontaneous glioma trial (U01CA224160-01). We found evidence of a strong effect of the therapy on peripheral immune cells in responder versus nonresponder dogs. Upon initiation of therapy, immune effector cell proliferation increased in responsive patients, whereas in nonresponders immune cell populations either didn’t change significantly or an increase in immunosuppressive phenotypes was observed. Cell populations such as MDSC, regulatory T cells and exhausted T cells were predominantly higher in nonresponder dogs. These data showed that peripheral immune cell phenotypes were linked to the outcome of the immunotherapy, thus dogs with better immune cell profiles, including increased cytotoxic differentiation, had a better prognosis and longer overall survival rates. Taken together, these results suggest that variation in patient responsiveness can be explained by differences in peripheral immune cell proliferation and differentiation soon after the initiation of immunotherapy.

Citation Format: Francisco W. Puerta Martinez, Sathya Schnell, Susan Arnold, Elizabeth G. Pluhar, Michael Olin. Circulating immune biomarkers in peripheral blood reflect the effect of a CD200 immune checkpoint targeted immunotherapy in pet dogs with high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1266.
Abstract 1289: Systemic immune response of prostate cancer patients to alpha-emitting radiotherapy
Authors: Lunj Sapna , Mistry Hitesh , Song YeePei , Patel Kamlesh , Nightingale Hannah , Smith Tim , Hoskin Peter , West Catharine , Choudhury Ananya
Published: June 2022

Targeted alpha therapy (TAT) is a type of radiotherapy (RT) that uses alpha-emitting radionuclides. These radionuclides have a natural affinity for areas of bone metastases (i.e., radium-223, Ra-223) or can label specific tumour targets (i.e., prostate-specific membrane antigen). Conventional photon irradiation (i.e., external beam) can activate or suppress immune responses. The resulting immune response is dictated by the release of soluble markers such as cytokines (i.e., IL-6) and damage associated molecular patterns, DAMPs (calreticulin). Less is known about the immune response induced by alpha-emitters. With increased interest in using TAT for cancer patients, it is important to gain a more in depth knowledge regarding the immune response of alpha emitters.

Aim: Explore the systemic immune response of prostate cancer (PCa) patients to alpha-emitting RT by studying circulating cytokines, checkpoint molecules, matrix metalloproteinases (MMPs) and DAMPs. Methods: The cohort comprised of 43 patients with metastatic castration-resistant PCa treated with up to six four-weekly injections of the alpha emitter, Ra-223. We measured longitudinal changes in 61 plasma markers using the Luminex assay. The plasma markers included cytokines, checkpoint molecules and DAMPs.

Results: Ra-223 induced longitudinal changes in 14 plasma markers over the course of treatment (T2-T6) compared with pre-treatment levels (T1). Most of the observed RT-induced changes occurred after four injections of Ra-223 (T5), where 9 of the 14 plasma markers changed significantly. The table summarises T5 findings (presented as mean concentration, pg/ml; statistical analysis using Wilcoxon matched-pairs signed rank test).

Conclusion: Our data demonstrates that alpha-emitting RT induces systemic immune responses that can be measured in the plasma of patients. Given the diverse role of these markers, further study is needed to explore the biological implications of these systemic changes on patient outcome.

Changes in plasma markers in patients treated with Ra-223 T1 T5 P value IL-8 6.7±0.9 9.7±1.3 0.0009 IL-2R 293.5±42.9 364.5±69.7 0.0247 IP-10 18.4±2.3 21.8±2.5 0.0243 RANTES 2668.8±173.4 2944.7±164.6 0.0455 BTLA 549.9±153.5 527.7±265.2 0.0319 CD137 49.6±8.9 39.1±13.5 0.0099 Calreticulin 7868±3514.9 9238.4±808.5 0.0386 RAGE 63.9±4.7 52.3±5.1 0.0014 MMP1 66.1±7.5 84.9±12.6 0.0245

Citation Format: Sapna Lunj, Hitesh Mistry, YeePei Song, Kamlesh Patel, Hannah Nightingale, Tim Smith, Peter Hoskin, Catharine West, Ananya Choudhury. Systemic immune response of prostate cancer patients to alpha-emitting radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1289.
Abstract 129: Effect of punicic acid on MCF7 breast cancer cells
Authors: Gbayisomore Oluwafemi , Klausner Hannah , Phelan Shelley A.
Published: June 2022

The pomegranate fruit has long been recognized for its health benefits in a wide range of chronic diseases. Components of pomegranate seed extracts (PSOs) have been shown to have antioxidant, anti-glycemic, and antimicrobial activity, and recent research has demonstrated that PSOs can inhibit the growth of select cancers in vitro. One of the main components of PSO is punicic acid, an omega-5 fatty acid that is capable of inhibiting breast and prostate cancer cell proliferation, although the mechanisms of action are not yet known. In order to further understand its potential anti-cancer properties, we investigated the effect of punicic acid on proliferation and viability of human MCF7 breast cancer cells. Cells were treated with various concentrations of punicic acid (between 10 and 100ug/ml) for up to 72 hours, and various endpoints were measured including cell proliferation, cytotoxicity, and apoptosis. We found that all doses led to a significant reduction in cell viability based on an MTS assay. We also found a dose-dependent effect of punicic acid on cytotoxicity, as measured by an LDH release assay. Cell death was confirmed to be, at least in part, due to apoptosis, as detected by Annexin V and propidium iodide fluorescence. While 10 ug/ml punicic acid induced measurable cell death by 48 hours, higher concentrations were toxic to cells within the first 24 hours of treatment. Since many of the peroxiredoxin (Prdx) family of antioxidant proteins are known to involved in cancer cell survival, and our lab previously demonstrated their regulation in MCF7 cells, we examined the effect of punicic acid on Prdx expression. We found members of this gene family to be significantly elevated within 24 hours of punicic acid treatment. Altogether, our data definitively demonstrates anti-cancer properties of punicic acid, as well as its ability to regulate antioxidant gene expression in these cells. This research has important implications in the field of natural products as therapeutic options for cancer treatment, as opposed to conventional chemotherapy.

Citation Format: Oluwafemi Gbayisomore, Hannah Klausner, Shelley A. Phelan. Effect of punicic acid on MCF7 breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 129.
Abstract 130: Pharmacological effects of selective xCT inhibition in ARID1A mutated cancer models
Authors: Henry Christophe , Péron Philippe , Blanchet Anne-Marie , Genevois-Borella Arielle , Trellu Marc , Bourges Dorothée , Lienard Philippe , Basset Alexandra , Jouannot Erwan , Philippo Christophe , Debussche Laurent , Moll Jürgen
Published: June 2022

xCT is a cystine/glutamate antiporter that exports intracellular glutamate for extracellular cystine. In the intracellular space, cystine is converted into cysteine which is subsequently used for glutathione (GSH) synthesis (a major antioxidant species). ARID1A is part of the SWI-SNF remodeling complex that binds on the xCT promoter and as such controls its gene expression. ARID1A deficiency which is highly prevalent in many cancers results in downregulation of xCT, impaired GSH biosynthesis and subsequent ROS induction, raising potential rationale to target xCT in ARID1A deficient cancers. By using a selective nanomolar range xCT inhibitor, we first showed that cell lines deficient for ARID1A were significantly more sensitive to the inhibition of xCT. Effects on proliferation could be reversed by N-acetylcysteine supplementation in the culture medium. In vivo, the xCT inhibitor confirmed its drug-like properties showing significant target engagement in an ARID1A deficient ovarian cell line xenograft model together with good PK and tolerability profiles. Altogether these studies have provided evidence that a specific drug-like inhibitor of xCT can be developed. This proprietary compound was used to confirm that in vitro the deficiency of ARID1A in ovarian cancer predicts sensitivity to xCT inhibition. Further in vivo pharmacological studies are required to confirm a specific molecular context predicting sensitivity towards xCT inhibition.

Citation Format: Christophe Henry, Philippe Péron, Anne-Marie Blanchet, Arielle Genevois-Borella, Marc Trellu, Dorothée Bourges, Philippe Lienard, Alexandra Basset, Erwan Jouannot, Christophe Philippo, Laurent Debussche, Jürgen Moll. Pharmacological effects of selective xCT inhibition in ARID1A mutated cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 130.
Abstract 1315: Tumor necrosis factor related apoptosis inducing ligand fosters myeloid derived suppressor cell mediated tumor immune evasion in cholangiocarcinoma
Authors: Loeuillard Emilien , Yang Jingchun , Dong Haidong , Gores Gregory J. , Ilyas Sumera I.
Published: June 2022

Background and Aims: Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is predominantly expressed on immune cells. Although TRAIL biology has garnered considerable attention as a potential anti-cancer strategy, TRAIL agonists have had very limited anti-cancer activity in humans. TRAIL signaling can attenuate the T lymphocyte response. However, this potential immunosuppressive function of TRAIL has not been examined in cancer biology. Cholangiocarcinoma (CCA), a highly lethal biliary tract cancer, has a dense immunosuppressive microenvironment. Accordingly, CCA provides a model to examine the potential immune regulatory function of TRAIL in cancer biology.

Methods: Using a syngeneic, orthotopic murine model of CCA (PMID: 29464042), murine CCA cells (SB cells) that express both Trail and Trail receptor (Tr) were implanted into livers of WT and Tr-/- mice. Hence, in this model the host immune cells express Trail but not the receptor; therefore, they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed, and tumors were characterized using flow cytometry.

Results: We observed that Tr-/- mice had a significant reduction in tumor burden compared to WT mice. Moreover, tumor bearing Tr-/- mice had a significant increase in cytotoxic T lymphocytes (CTLs) and enhanced CTL effector function. Myeloid-derived suppressor cells (MDSCs) were significantly decreased in Tr-/- tumors compared to WT tumors. Furthermore, implantation of SB cells devoid of Trail (SB-Trail-/-) into WT mice resulted in a significant reduction in tumor burden and MDSC infiltration. In vitro functional studies employing SB cells deficient in Tr (SB-Tr-/-) and MDSCs were carried out. There was a significant reduction in proliferation and immunosuppressive function of MDSCs when cocultured with SB-Tr-/- compared to SB cells. These results indicate that TRAIL-TR fosters MDSC growth and immunosuppressive function.

In conclusion, we have demonstrated that Tr-/- mice have a significant reduction in CCA tumor burden and MDSC infiltration. Consequently, Tr-/- tumors have enhanced CTL infiltration and function. These data suggest that the TRAIL-TR system mediates tumors immune evasion via MDSCs, and direct targeting of TRAIL on CCA cells is a potential anti-tumor strategy.

Citation Format: Emilien Loeuillard, Jingchun Yang, Haidong Dong, Gregory J. Gores, Sumera I. Ilyas. Tumor necrosis factor related apoptosis inducing ligand fosters myeloid derived suppressor cell mediated tumor immune evasion in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1315.
Abstract 1344: Immune transcriptional profiling in a large animal model of histiocytic sarcoma reveals putative drivers and tumor suppressors
Authors: Lenz Jennifer A. , Assenmacher Charles-Antoine , Radaelli Enrico , Atherton Matthew J.
Published: June 2022

Histiocytic sarcoma (HS) is a rare, yet lethal malignancy with a median overall survival of six months and no universally agreed standard of care therapy. Due to the paucity of human cases, there is a drive to develop model systems to improve our understanding of the pathology underlying this disease and screen new treatment strategies. Spontaneous canine HS exhibits increased prevalence in specific breeds, shares key genetic and biologic similarities with the human disease, and occurs in an immunocompetent setting. Our previous studies revealed a spectrum of hot and cold immune tumor microenvironments (TME) based on tumor infiltrating lymphocyte (TIL) quantification in 18 canine and 5 human HS cases. In dogs, increased TIL densities were positively associated with longer survival times. Despite this association, the disease course of HS in our veterinary patient cohort was nearly uniformly fatal. To evaluate for potential inhibitors of T cell immunity in the TME and identify putative drivers and suppressors of HS, we performed immune transcription profiling. Tumors were selected from 3 short-lived cases of splenic HS that were sparsely infiltrated with TIL and 3 long-lived cases of pulmonary HS that were T cell inflamed. Tumors were compared to corresponding grossly healthy splenic and lung tissues collected from 3 dogs without neoplasia undergoing routine post-mortem examination. RNA was extracted from formalin-fixed paraffin embedded tissues and immune transcription profiling was performed using the NanoString nCounter Canine IO Panel. Analyses of all tumors compared to normal tissues, and subset analyses of tissues stratified based on tissue origin site, revealed enrichment of ITGAX (CD11c) expression in the HS tumor samples consistent with histiocytic origin. In each subset analysis, SPP1 (osteopontin) was highly expressed in HS tumors compared with normal tissues. Osteopontin has been shown to be protumorigenic in a variety of cancers, is a potent chemokine for macrophages, and can suppress T cell activation through ligation of CD44, thus acting as a T cell checkpoint. Additionally, we found decreased expression of TXNIP in HS tumors compared to healthy tissues. TXNIP is an established tumor suppressor gene in various tumor types, including acute myeloid leukemia. Future studies will interrogate the functional implications of these transcriptional changes with the aim of developing novel therapeutic strategies for HS in both species.

Citation Format: Jennifer A. Lenz, Charles-Antoine Assenmacher, Enrico Radaelli, Matthew J. Atherton. Immune transcriptional profiling in a large animal model of histiocytic sarcoma reveals putative drivers and tumor suppressors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1344.