bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3710: Synergistic cell death induction in ovarian cancer by cisplatin and ABT-199 is mediated by expression of NOXA
Authors: Schaefer Benjamin , Weller Sandra , Beigl Tobias B. , Schulze-Osthoff Klaus , Kopp Hans-Georg , Aulitzky Walter E. , Essmann Frank
Published: June 2022

Ovarian cancer is among the top ten leading causes of cancer death in women worldwide with a mortality rate of roughly 5% among all cancer deaths. So far, there are no recommended screening tests for the early detection of ovarian cancer. Therefore, patients are frequently diagnosed at late stages of ovarian cancer, which is associated with unfavorable 5-year survival. Patients suffering from ovarian cancer often show a good response to initial therapy with platinum-based compounds and/or taxanes subsequent to resection of the affected tissue. Unfortunately, ovarian cancer shows a high frequency of relapse and after chemotherapy persistent tumor cells re-expand and successively evolve into therapy-resistant tumors.

We recently reported that cisplatin (cisPt) treatment of ovarian cancer cells enhances mitochondrial content and mitochondrial reactive oxygen species (Kleih et al. 2019). We therefore speculated that the enhanced mitochondrial content primes cisPt-treated cells for apoptosis induced by BH3 mimetic drugs, such as Venetoclax (ABT-199), that act on anti-apoptotic Bcl-2 proteins at the outer mitochondrial membrane. To explore this hypothesis, we investigated whether the combined administration of cisPt and ABT-199 leads to a synergistic cell death induction in ovarian cancer cells with different cisPt resistance and mitochondrial content.

Detection of cell death by flow cytometric measurements of the mitochondrial membrane potential and exposure of phosphatidylserine demonstrated a synergistic cell death induction by cisPt and ABT-199 in OVCAR4 and OVCAR8 cells, irrespective of the cisPt resistance. Interestingly, Western blot analysis revealed that ABT-199 induced the accumulation of the BH3-only protein NOXA in both cancer cell lines. Accumulation of NOXA was dependent on ABT-199-mediated activation of the stress-responsive transcription factor ATF4. Knock-down experiments showed that apoptosis of cisPt-resistant OVCAR8 cells by the combined treatment with ABT-199 and cisPt was entirely dependent on NOXA. Moreover, also cisPt-sensitive OVCAR4 cells revealed an initially reduced apoptosis induction by the NOXA knock-down.

Our data therefore elucidate the molecular mechanism of the synergistic efficacy of cisPt/ABT-199 combination therapies, which should be promising especially for the treatment of recurrent tumors developing reduced sensitivity to cisPt-based treatments.

Citation Format: Benjamin Schaefer, Sandra Weller, Tobias B. Beigl, Klaus Schulze-Osthoff, Hans-Georg Kopp, Walter E. Aulitzky, Frank Essmann. Synergistic cell death induction in ovarian cancer by cisplatin and ABT-199 is mediated by expression of NOXA [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 3710.
Abstract 3711: Modulation and inhibition of prostate cancer cells by microbial metabolites and resveratrol
Authors: Stokes James , Singh Rajesh , Mishra Manoj K.
Published: June 2022

The chemotherapeutic compounds resveratrol (RES) and short-chain fatty acids (SCFAs) are potent inhibitors of cancer cell proliferation during in vitro testing. These compounds are naturally occurring and present relatively low toxicity to normal cells and tissues, making them an attractive cancer treatment alternative. SCFAs are the chemical by-products of microbial activity known as fermentation. RES is a polyphenolic compound found in select fruits and vegetables and has been known for its versatility in preventing and reversing many diseases, including several cancers. Butyric and propionic acid are considered the most potent of the SCFAs found within the intestinal lumen. The prime goal of this research is to understand if the RES and SCFA compounds use a shared biological pathway(s) to fight and inhibit cancer. We used TRAMP C1 and C3 prostate cancer cells (transgenic adenocarcinoma mouse prostate) as an experimental model. These cells were treated with the RES and SCFAs both singularly and synergistically for 24hrs. The cells were analyzed to understand the potential cell toxicity and proliferation, the modulation of apoptotic and proapoptotic proteins/genes, and any change in mitochondrial membrane potential pathways. The result demonstrated that the expression of apoptogenic proteins induces programmed cell death in a mitochondria-independent manner. Therefore, the result suggests that RES and SCFAs can facilitate many critical biological functions within the host's body, including cellular respiration, nutrient assimilation, and various immunological responses to inhibit cancer cell proliferation.

Citation Format: James Stokes, Rajesh Singh, Manoj K. Mishra. Modulation and inhibition of prostate cancer cells by microbial metabolites and resveratrol [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 3711.
Abstract 3724: PBRM1 loss in RCC: Implications for immunotherapy
Authors: Metz Alexander , Sikder Rahmat , Ellithi Moataz , Slifker Micheal , Abbosh Philip
Published: June 2022

Introduction The association between mutational status of the polybromo-1 (PBRM1) gene in clear cell renal cell carcinomas (ccRCCs) and papillary RCCs (pRCCs) in the response to immune checkpoint blockade is debatable. Here, we aim to characterize the biological changes that PBRM1 loss causes and how these changes might modulate the immune response to cancer.

Methods and Results Gene set enrichment analysis (GSEA) of PBRM1-mutant RCC tumors from TCGA data sets reveals NF-kB signaling via TNFα, hypoxia, and p53 signaling pathways are enriched compared to PBRM1 WT. PBRM1-mutant tumors also have increased number of insertion/deletion mutations and non-synonymous mutations that are directly proportional to neoantigen burden. CIBERSORT analysis of PBRM1-mutant ccRCC tumors reveals decreased transcriptional signature associated with CD8+ T-cell infiltration and lower levels of two cytolytic signatures. PBRM1-mutant ccRCC cell lines have constitutively activated NF-kB signaling, as well as a greater induction of NF-kB in response to TNFα via western blot when compared with PBRM1 reconstituted cells. Additionally, PD-L1 is more strongly expressed on the cell surface of PBRM1-mutant cells, which may modulate their interactions with the immune system. However, MHC-I expression was not found to be significantly different between PBRM1 mutant and reconstituted cells.

Conclusions These results shed light on changes in biology and immunogenicity associated with or caused by PBRM1 loss. They also implicate NF-kB as a potential moderator of immune response, which should be studied further to characterize its effects on immune checkpoint blockade therapy (ICB).

Citation Format: Alexander Metz, Rahmat Sikder, Moataz Ellithi, Micheal Slifker, Philip Abbosh. PBRM1 loss in RCC: Implications for immunotherapy [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 3724.
Abstract 3726: Functional genomic dissection of PIK3CA hotspot mutations using a multi-lineage isogenic breast cell model
Authors: Miranda Adam X. , Hodges Emily , Park Ben Ho
Published: June 2022

PIK3CA is the most commonly mutated gene in breast cancer, and in breast cancer there are two hotspot mutations that represent an outsized proportion of PIK3CA mutations. Previous research suggests that there are distinct prognostic and cell signaling differences induced by these mutational isoforms, however there is currently only one targeted treatment approved for PIK3CA mutant breast cancer. The goal of our research is to leverage a comprehensive multi-lineage isogenic breast cell line model to further interrogate the unique effects of these PIK3CA mutations on breast cells using assays to assess gene expression and chromatin state. Insights gained from these assays shed light on disrupted pathways that may prove to be new targets for PIK3CA mutation-specific treatment of breast cancer.

Citation Format: Adam X. Miranda, Emily Hodges, Ben Ho Park. Functional genomic dissection of PIK3CA hotspot mutations using a multi-lineage isogenic breast cell model [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 3726.
Abstract 3735: Regulation of HPV gene expression by 5-azacytidine treatment in head and neck cancer
Authors: Rehmani Hina , Isaeva Natalia , Yarbrough Wendell G.
Published: June 2022

Human papillomavirus (HPV) causes approximately 80% of head and neck squamous cell carcinoma (HNSCC) in the oropharynx, and its incidence is now higher than uterine cervical cancer in the US. The CDC estimates that this common sexually transmitted disease accounts for nearly 13,000 HPV+ HNSCC patients per year in the United States. Despite having a higher cure rate than HPV- HNSCC, the recurrence rate is approximately 30%. These patients have few therapeutic options since resistance to treatment arises from all known therapeutic modalities. Initial therapy for HPV+ HNSCC is aggressive and often results in negative lifelong side effects. The substantial recurrence rate and high morbidity of primary therapy suggest the need for new treatments. Global genome hypermethylation in HPV+ HNSCC, compared to HPV-, prompted our lab to explore demethylation as a therapeutic option. 5-azacytidine (5-aza) and 5-aza-2′-deoxycytidine (decitabine) are synthetic cytidine analogs that cause DNA demethylation by trapping methyltransferases to chromatin. These demethylating agents are FDA approved for the treatment of myelodysplasia and acute myeloid leukemia. Recently, we found that HPV+ HNSCC cells are more sensitive than HPV- cells to 5-aza, that low doses of 5-aza delayed HPV+ xenograft tumor growth, and that HPV+ tumor samples from patients treated with 5-aza in a window trial had increased apoptosis. We also observed a marked downregulation of HPV gene expression after 5-aza. Decreased HPV E6 expression, followed by reactivation of tumor suppressor p53, induced p53-dependent apoptosis in HPV+ HNSCC. Thus, decreased expression of HPV genes is an important component of 5-aza-associated toxicity toward HPV+ HNSCC; therefore, we further explored mechanisms through which demethylation negatively regulates the expression of HPV genes. First, we determined that 5-aza regulates transcription of HPV genes, but not the stability of HPV transcripts. Second, we found that transcriptional regulation of HPV genes after 5-aza depends on the proto-oncogene JunB, a component of the transcription factor Activator Protein 1 (AP-1). Moreover, our experiments revealed that clonogenic survival of HPV+ head and neck cancer cells is dependent on JunB expression. In summary, our study provides an improved mechanistic understanding of the regulation of HPV gene transcription in HNSCC and identifies a novel HPV+ HNSCC dependency on JunB, potentially providing a basis for a new rational targeted therapy.

Citation Format: Hina Rehmani, Natalia Isaeva, Wendell G. Yarbrough. Regulation of HPV gene expression by 5-azacytidine treatment in head and neck 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 3735.
Abstract 3743: Genome-wide profiling of DNA N6-methylation from a breast cancer and a matched normal cell lines
Authors: Xiao Chunlin , Schneider Valerie
Published: June 2022

DNA N6-methyladenine (m6A) modification has been found widely presented in the human genome, and genome-wide DNA methylation profiling in cancer may reveal epigenetic signatures with significant clinical outcomes. Whole genome sequencing data from PacBio single-molecule real-time (SMRT) system provides signals for identifying the presence of m6A in human genomic DNA. We identified 343,199 m6A modification sites with an average density of 122 per Mb in the HCC1395 breast cancer cell line, whereas 722,303 m6A modification sites with average density of 257 per Mb were observed in a matched normal HCC1395BL cell line, meaning that the total number and the average density of m6A methylation sites in the cancer cell line were reduced more than 50% than that in normal cell line. Only small fraction of the methylation sites was found to be shared between the two cell lines, indicating that significant de-methylation (loss) and new methylation (gain) events occurred in HCC1395 cancer cells. A broad distribution of m6A methylation sites across autosomal chromosomes was observed, but the density of m6A methylation sites on chromosome X was extremely low for both HCC1395 and HCC1395BL cell lines. In contrast, the m6A densities on chromosome 7 and chromosome 22, particularly on their q-arms, from HCC1395 were substantially higher (hypermethylation) than other autosomal chromosomes, suggesting that copy number variations may be associated with these chromosomal regions. Most of the m6A methylation sites were located in intergenic and intronic regions, whereas only about 2~3% of the m6A methylation sites were situated in exonic regions, and 12% on ncRNAs. Understanding the genome-wide distinction of DNA methylation between cancer and matched normal cell lines makes it possible to ask more targeted questions and further investigate the role of methylation in cancer.

Citation Format: Chunlin Xiao, Valerie Schneider. Genome-wide profiling of DNA N6-methylation from a breast cancer and a matched normal cell lines [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 3743.
Abstract 3817: A single-cell, spatial multiomics atlas and cellular interactome of all major skin cancer types
Authors: Grice Laura , Ni Guiyan , Jin Xinnan , Tran Minh , Killingbeck Emily , Gregory Mark , Mulay Onkar , Teoh Siok-Min , Kulasinghe Arutha , Leon Michael , Murphy Sarah , Warren Sarah , Kim Youngmi , Nguyen Quan
Published: June 2022

Skin cancer is by far the most common cancer, encompassing squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and melanoma. The diversity of cell types and tissue organisation in skin cancer remains poorly understood yet is required to improve diagnosis and treatment. In this work, we integrated six imaging and sequencing technologies to build the first spatial single cell reference for all three major skin cancer types and create a comprehensive skin cancer interactome. Using single-cell RNA-Seq (RNA) of >100,000 cells from 11 paired patient biopsies, we identified 28 SCC cell types, including 10 immune cell types, and found core suites of 39 cancer genes and 222 healthy genes shared across ≥80% patient samples. Using independent Nanostring Digital Spatial Profiling (RNA, protein), we validated most immune cell types and gene markers at protein and RNA levels. The enrichment of an immune signalling signature in SCC was further revealed by spatial Nanostring Single Molecular Imaging - SMI (RNA). Strikingly, we found the high consistency in mapping cell types in scRNAseq data and the independent SMI data, for example, the distribution of the three keratinocyte layers (basal, cycling and differentiated). This observation suggested the power of combining scRNAseq data with spatial SMI data. Furthermore, we implemented three approaches to validate the spatial distribution and cell type co-localisation by both Visium Spatial Transcriptomics (RNA), SMI (RNA) and Opal Multiplex Polaris (protein). Finally, cell-cell interactions were inferred at the global level using scRNAseq data (no spatial information) and Visium data (with spatial dimension), which were then validated at high throughput (517 ligands/receptors) and single-cell resolution using SMI. These in situ interaction maps were built across all three cancer types to create a comprehensive spatial interaction atlas of skin cancer. We also used targeted approaches with Polaris (protein) and RNAScope (RNA) to confirm and visualise clinically-important ligand-receptor pairs, including checkpoint inhibitor drug targets PD-1 and PD-L1. By integrating six distinct yet complementary spatial and single cell technologies, this study highlights the power of a spatial multi-omics approach for understanding cell types and their activities in cancer tissues.

Citation Format: Laura Grice, Guiyan Ni, Xinnan Jin, Minh Tran, Emily Killingbeck, Mark Gregory, Onkar Mulay, Siok-Min Teoh, Arutha Kulasinghe, Michael Leon, Sarah Murphy, Sarah Warren, Youngmi Kim, Quan Nguyen. A single-cell, spatial multiomics atlas and cellular interactome of all major skin cancer types [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 3817.
Abstract 3825: Germline modifiers of the tumor immune microenvironment reveal drivers of immunotherapy response
Authors: Pagadala Meghana , Wu Victoria , Pérez-Guijarro Eva , Kim Hyo , Castro Andrea , Talwar James , Gonzalez-Colin Cristian , Cao Steven , Schmiedel Benjamin J. , Sears Timothy , Goudarzi Shervin , Kirani Divya , Salem Rany M. , Morris Gerald P. , Harismendy Olivier , Patel Sandip P. , Mesirov Jill P. , Zanetti Maurizio , Day Chi-Ping , Fan Chun C. , Thompson Wesley K. , Merlino Glenn , Gutkind J. Silvio , Vijayanand Pandurangan , Carter Hannah
Published: June 2022

With the continued promise of immunotherapy as an avenue for treating cancer, understanding how host genetics contributes to the tumor immune microenvironment (TIME) is essential to tailoring cancer risk screening and treatment strategies. Using genotypes from over 8,000 European individuals in The Cancer Genome Atlas and 137 heritable tumor immune phenotype components (IP components), we identified and investigated 532 TIME-SNPs. Focusing on 77 variants that were relevant to cancer risk, survival, or treatment response, we explored their potential to reveal novel targets for immunotherapy. Many variants overlapped regions with histone marks indicating active transcription, and influenced gene activities in specific immune cell subsets, such as macrophages and dendritic cells. TIME-SNPs implicated genes such as LAIR1, TREX1, CTSS, CTSW and LILRB2 were differentially expressed between responders and non-responders to immune-checkpoint blockade (ICB) in preclinical studies. Of these, LILRB2 and LAIR1 have already been identified as putative targets for immunotherapy. Here we found that inhibition of CTSS led to better tumor control and survival in murine models, alone or in combination with anti-PD-1. Collectively we show that through an integrative approach, it is possible to link host genetics to TIME characteristics, informing novel biomarkers for cancer risk and target identification in immunotherapy.

Citation Format: Meghana Pagadala, Victoria Wu, Eva Pérez-Guijarro, Hyo Kim, Andrea Castro, James Talwar, Cristian Gonzalez-Colin, Steven Cao, Benjamin J. Schmiedel, Timothy Sears, Shervin Goudarzi, Divya Kirani, Rany M. Salem, Gerald P. Morris, Olivier Harismendy, Sandip P. Patel, Jill P. Mesirov, Maurizio Zanetti, Chi-Ping Day, Chun C. Fan, Wesley K. Thompson, Glenn Merlino, J. Silvio Gutkind, Pandurangan Vijayanand, Hannah Carter. Germline modifiers of the tumor immune microenvironment reveal drivers of immunotherapy response [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 3825.
Abstract 383: ONCR-021 as a systemic intravenous synthetic RNA virus immunotherapy for the repeat treatment of cancer
Authors: Bryant Jeffrey , Denslow Agnieszka , Hewett Jacqueline , Cong Lingxin , De Almeida Ana , Lee Jennifer , Jacques Judy , Feau Sonia , Wambua Daniel. , Yanez Adrienne , Wang Pam Shou-Ping , Deterling Jessica , Scott Matthew , Auer Jason , Haines Brian B. , Quéva Christophe , Lerner Lorena , Kennedy Edward M.
Published: June 2022

Oncolytic viruses (OV) have shown great potential to improve clinical outcomes when dosed intratumorally, however, their therapeutic efficacy when intravenously administered is likely limited by the rapid emergence of neutralizing antibodies. To overcome this limitation, we developed Synthetic RNA viruses consisting of a replication competent viral genomic RNA (vRNA) encapsulated within a lipid nanoparticle (LNP) for IV administration. Upon dosing and delivery of this infectious RNA payload, the vRNA initiates viral replication and virus production in neoplastic cells leading to oncolysis and tumor destruction. This formulation enables repeat intravenous dosing of a replication competent oncolytic virotherapy even in presence of circulating neutralizing antibody to the virus. Here we present ONCR-021, an LNP formulation of Coxsackievirus A21 (CVA21) vRNA. ONCR-021 vRNA encodes a novel ICAM1-dependent strain of CVA21 that results in greater in vitro and in vivo oncolysis compared to the previously described CVA21 Kuykendall strain. ONCR-021 is broadly oncolytic in cancer cell lines in vitro and is intended for clinical development in NSCLC, RCC, and HCC based upon the viral tropism. IV-administration of ONCR-021 vRNA results in rapid initiation of viral replication, oncolysis, and potent anti-tumor efficacy, even in the presence of circulating CVA21 neutralizing antibodies. This efficacy is principally driven by CVA21 amplification in situ after delivery to tumor cells and we demonstrate viral replication, virion production and spread within the tumor after dosing. We also observe only modest and transient production of CVA21 in healthy tissues of transgenic mice expressing the CVA21 entry receptor human ICAM1. Consistent with these findings, high doses levels of ONCR-021 were well-tolerated in this model. Altogether, these preclinical data support the development of ONCR-021, a novel synthetic oncolytic virus designed to overcome the challenges of repeat intravenous administration of viral immunotherapy for the treatment of disseminated cancers.

Citation Format: Jeffrey Bryant, Agnieszka Denslow, Jacqueline Hewett, Lingxin Cong, Ana De Almeida, Jennifer Lee, Judy Jacques, Sonia Feau, Daniel. Wambua, Adrienne Yanez, Pam Shou-Ping Wang, Jessica Deterling, Matthew Scott, Jason Auer, Brian B. Haines, Christophe Quéva, Lorena Lerner, Edward M. Kennedy. ONCR-021 as a systemic intravenous synthetic RNA virus immunotherapy for the repeat treatment of 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 383.
Abstract 3835: Kindlin-2 complexes containing α6β1 integrin are responsive to hypoxia
Authors: Hernandez-Cortes Daniel , Gard Jaime M.C. , Knudsen Beatrice S. , Warfel Noel A. , Cress Anne E.
Published: June 2022

The laminin-binding integrins are mechanosensory receptors critical for cell adhesion and structural organization that link the extracellular matrix (ECM) to the cytoskeleton. Integrin α6β1 is associated with prostate cancer (PCa) migration, invasion, metastasis, and decreased cancer-specific survival. Kindlin-2 (FERMT2) is a β1 integrin adaptor and mechanosensory focal adhesion (FA) protein that activates and clusters integrins in response to structural ECM alterations in the tumor microenvironment. Our goal was to determine if integrin-kindlin-2 adhesion complexes (kindlin-2:α6β1) were responsive to hypoxia, a physiologically relevant and altered microenvironment in PCa progression. Five different endpoints were tested including the biochemical analysis of kindlin-2 complexes, qRT-PCR, immunoblotting, immunocytochemistry, and electric cell impedance sensing (ECIS). Using DU145 prostate cancer cells grown under hypoxia (1% O2) for up to 16 hours, the results showed a reversible increase in kindlin-2:α6β1 complexes with maximal assembly within 4 hours and disassembly starting by 8 hours. Notably, kindlin-2:α6β1 complexes were found exclusively within membrane projections and were not observed within hypoxia-inducible paxillin (PXN)-containing FAs. The hypoxia induced kindlin-2:α6β1 complexes and classical FAs were dependent on kindlin-2 as determined by CRISPR-Cas9 heterozygous deletion of FERMT2. Protein co-localization of α6 integrin and PXN with kindlin-2 within membrane projections and FAs, respectively, was also induced under hypoxia. Further, non-invasive ECIS measurements in live cells confirmed functional cell-cell and cell-ECM dynamics driven by hypoxia and requiring kindlin-2. Our results indicate that the kindlin-2:α6β1 complexes are uniquely associated with FA-independent membrane projections induced by hypoxia, a tumor microenvironment associated with aggressive prostate cancer. The novel kindlin-2:α6β1 complexes may represent an actionable pharmacological target for blocking escape of organ confined disease and metastasis promoting steps of human prostate cancer.

(Partially supported by NIH grants CA P30 23074, DOD W81XWH-19-1-0455, and NCI R01 CA242226).

Citation Format: Daniel Hernandez-Cortes, Jaime M.C. Gard, Beatrice S. Knudsen, Noel A. Warfel, Anne E. Cress. Kindlin-2 complexes containing α6β1 integrin are responsive to hypoxia [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 3835.
Abstract 3836: Mapping the interactome of the endogenous metalloproteinase inhibitor TIMP2 using extracellular proximity labeling (ePL)
Authors: Peeney David , Gurung Sadeechya , Liu Yueqin , Lazaroff Carolyn , Richie Christopher , Stetler-Stevenson William G.
Published: June 2022

Tissue inhibitors of metalloproteinases (TIMPs) are a family of endogenous proteins that were initially defined by their ability to inhibit the enzymatic activity of matrix metalloproteinases (MMPs), the major mediators of ECM breakdown and turnover. TIMPs are important regulators of ECM structure, function and homeostasis. Since their original discovery, additional MMP-independent functions have been attributed to TIMP family members leading to their designation as multifunctional proteins with discrete functional domains. TIMP2 is a unique family member, with a broad expression profile that is expressed in both normal and diseased tissues, even in those with minimal metalloproteinase activity. Understanding the functional transformation of matrisome regulators, like TIMP-2, during the dynamic evolution of tissue microenvironments associated with disease progression is essential to the development of ECM targeted therapeutics. This knowledge may also garner understanding of therapeutic resistance and the failure of conventional and next-generation cancer therapies. Interactome studies are predominantly performed in non-living systems, and very rarely performed in complex culture systems such as 3D spheroids/co-cultures. To investigate the TIMP-2 interactome in complex biological systems we recently developed carboxyl- and amino-terminal fusion genes of TIMP-2 with the promiscuous biotinylating peptides BioID2 and TurboID. Packaged for retroviral delivery, this system gives us great flexibility in assessing the TIMP-2 interactome in simple versus complex 3D co-culture systems. We show, for the first time, that conditions can be optimized to support proximity labeling reactions in the extracellular milieu, representing an important technical resource for extracellular matrix biologists. Using this method, we identify novel interacting partners for TIMP-2 that provide insight into the tissue-specific actions of this protein in both healthy and diseased tissues.

Citation Format: David Peeney, Sadeechya Gurung, Yueqin Liu, Carolyn Lazaroff, Christopher Richie, William G. Stetler-Stevenson. Mapping the interactome of the endogenous metalloproteinase inhibitor TIMP2 using extracellular proximity labeling (ePL) [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 3836.
Abstract 3857: A novel microfluidic platform for understanding the role of PDAC stroma in immune response
Authors: Geyer Marlene , D'Agosto Sabrina , Lanz Henriëtte , Corbo Vincenzo , Joore Jos , Queiroz Karla S.
Published: June 2022

Pancreatic Ductal Adenocarcinoma (PDAC), the most common pancreatic cancer type is estimated to become the second leading cause of cancer-related deaths by 2030 with mortality rates of up to 93%. PDAC is characterized by high-density stroma and an immunosuppressive tumor microenvironment that lead to a lack of efficacy of immunotherapeutic strategies. Those facts highlight the urgent need for more relevant PDAC in vitro models that support the discovery of novel (immuno-)therapies. Here, we describe the establishment of a multiple cell type culture in a microfluidic platform that will be applied for modeling immune and stroma cell interplay in PDAC. Microfluidic platforms such as the OrganoPlate® support 3D growth and inclusion of different cell types cultured under flow conditions. The aim of this project was to determine the involvement of stromal cells such as endothelial cells and pancreatic stellate cells in immune cell recruitment in pancreatic cancer. In order to generate a more relevant culture set up, supporting cells and PDAC organoids were used in the 3-lane OrganoPlate® to study immune cell recruitment. Migration of fluorescently labelled peripheral blood mononuclear cells from vasculature through the stromal compartment to the PDAC organoids was tracked for 72h using confocal microscopy. Migration of immune cells to the tumor cell compartment was significantly inhibited by the stromal compartment, thus confirming in vivo immune responses, as the stroma seems to form both a physical as well as a chemical barrier for immune cells to reach the active tumor site. Interestingly, PDAC organoid conditioned medium efficiently attracts immune cells into the tumor compartment, indicating that secreted factors play a relevant role in immune cell recruitment. Despite the positive effect on recruitment and immune-tumor cell interaction, a lack of tumor cell killing is observed. The co-culture of PDAC organoids with stromal and immune cells grown on-a-Chip as described in this study indicates the suitability of microfluidic platforms for generating complex models and its use for dissecting complex cellular interplay and processes involved in the lack of anti-tumor immune responses in PDAC.

Citation Format: Marlene Geyer, Sabrina D'Agosto, Henriëtte Lanz, Vincenzo Corbo, Jos Joore, Karla S. Queiroz. A novel microfluidic platform for understanding the role of PDAC stroma in immune response [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 3857.
Abstract 3871: Solid tumor analytical validation of a T-regulatory immunohistochemistry multiplex for clinical studies
Authors: Gérus-Durand Marie , Baranova Elena , Tliba Maroua , Iglesias Sabine , Motte Manon , Burrer Renaud , Finan-Marchi Amanda
Published: June 2022

While classic, chromogenic immunohistochemistry (IHC) remains a mainstay in clinical diagnostics, it can be limiting in the era of immuno-oncology. In the field of immuno-oncology, where access to tissue samples may be limited, there is a need for an accurate interpretation of the sociology and in-depth phenotyping of the cells in the tumor microenvironment that requires a higher level of targets to investigate. Cytotoxic T cells are the main effector cells against tumors. Conversely, T-regulatory cells play a more pro-tumor role by suppressing cytotoxic T cell activation. The importance of the balance and tumor infiltration of these two cell populations has repeatedly been demonstrated in the literature. Due to this, Cerba Research was driven to develop an immunofluorescent multiplex to allow cytotoxic and regulatory T cells detection on the same tissue section for use in solid tumor clinical trials. Following our standard workflow, we developed a CD3-CD8-FoxP3 IHC multiplex (Histoprofile®- T-reg light). This panel allows for analysis of cytotoxic T-cells (CD3+/CD8+/FoxP3-), T helper cells (CD3+/CD8-/FoxP3-) and T regulatory cells (CD3+/CD8-/FoxP3+). Then following our standard process, we performed an analytical validation on a range of formalin fixed paraffin embedded solid tumors (including but not limited to Non Small Cell Lung Cancer, Head and Neck Squamous Cell Carcinoma, Colorectal Cancer and Breast Cancer). Specificity was verified by a pathologist on control tonsil tissue. The robustness of the protocol on a range of solid tumors was verified by the pathologist on tissue blocks and tumor microarrays. Precision tests (intra- and inter-assay) demonstrated a high level of panel reproducibility in solid tumor tissues - CD3 (CV 21.24%), CD8 (CV 19.5%), FoxP3 (CV 13.13%). Using Halo® software (Indica Labs), spatial analysis of the populations of interest can be appreciated in the tumor and stroma compartments. Cerba Research’s Histoprofile®- T-reg light panel passed all the analytical validation criteria for use in clinical studies. Cerba Research can provide this off the shelf panel in solid tumor clinical studies, allowing an elevated analysis of multiple immune biomarkers on a single tissue slide.

Citation Format: Marie Gérus-Durand, Elena Baranova, Maroua Tliba, Sabine Iglesias, Manon Motte, Renaud Burrer, Amanda Finan-Marchi. Solid tumor analytical validation of a T-regulatory immunohistochemistry multiplex for clinical studies [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 3871.
Abstract 3872: Overexpression of miR-200 in cancer-associated fibroblasts suppresses the migration and invasion of cancer cells in co-culture systems
Authors: Cheon Inyoung , Lee Sieun , Ahn Young-Ho
Published: June 2022

Cancer-associated fibroblasts (CAFs) promote cancer progression and metastasis in the tumor microenvironment. CAFs are regulated by diverse processes including regulation by microRNAs (miRNAs). To profile miRNAs involved in the regulation of CAF activation, we performed the NanoString nCounter® assay in mouse CAFs and normal lung fibroblasts (LFs). Among the miRNAs differentially expressed between CAFs and LFs, miR-200 family members were significantly downregulated in CAFs. Overexpression of miR-200 in CAFs suppressed the migration and invasion of lung cancer cells in co-culture systems (transwell migration and spheroid invasion assays). Next, we tried to identify novel miR-200 target genes based on RNA sequencing data obtained from control and miR-200-overexpressed CAFs, lung adenocarcinoma TCGA data, and TargetScan. We are now studying on these candidates to see whether they are involved in the interaction between CAFs and cancer cells.On the basis of these findings, we conclude that miR-200 suppresses the interaction between CAFs and lung cancer cells in the tumor microenvironment.

Keywords: cancer-associated fibroblasts, miR-200, lung cancer, tumor microenvironment, invasion

Citation Format: Inyoung Cheon, Sieun Lee, Young-Ho Ahn. Overexpression of miR-200 in cancer-associated fibroblasts suppresses the migration and invasion of cancer cells in co-culture systems [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 3872.
Abstract 3945: Novel antagonists of TEAD palmitoylation inhibit the growth of Hippo-altered cancers in preclinical models
Authors: Kamal Adeela , Candi Aurélie , Versele Matthias , Vanderhoydonck Bart , Marchand Arnaud , de Jong Ron , Hoang Thuy , Halder Georg , Chaltin Patrick , Gwaltney Stephen L. , Burgess Mike
Published: June 2022

Background: The Hippo pathway is an evolutionarily conserved signaling cascade whose deregulation can promote excessive cell proliferation and tumor development. Pathway output is mediated by the YAP and TAZ transcriptional co-activators, which bind to TEAD family transcription factors to drive target gene expression. Genomic aberrations in Hippo pathway components result in constitutive activation of YAP/TAZ, as seen with NF2 mutations in subsets of mesothelioma and other cancers. Hyperactivation of YAP/TAZ has also been associated with resistance to a variety of targeted agents, including EGFR and CDK4/6 inhibitors, suggesting that targeting the pathway may have utility as part of rationally selected combinations, in addition to genomically-informed monotherapy applications. Activity of the YAP/TAZ-TEAD complex thus represents a compelling pharmacologic target, due to its essential role in the pathway, and the presence of a conserved druggable site in TEAD that is required for transcriptional function.

Results and Discussion: Using biophysical techniques, we identified novel small molecules that bind to the TEAD auto-palmitoylation pocket. Initial hits were optimized for antagonism of TEAD-based transcription and drug-like properties, ultimately producing highly potent and orally bioavailable TEAD inhibitors. These compounds selectively inhibited the proliferation of cancer cell lines harboring genomic alterations in the Hippo pathway with low nM potency. In vivo models of Hippo pathway-altered xenografts showed consistent monotherapy activity, with dose-dependent and durable tumor regressions achieved at well-tolerated doses. Further characterization of these compounds as monotherapies and as part of rationally-designed combination regimens is ongoing.

Citation Format: Adeela Kamal, Aurélie Candi, Matthias Versele, Bart Vanderhoydonck, Arnaud Marchand, Ron de Jong, Thuy Hoang, Georg Halder, Patrick Chaltin, Stephen L. Gwaltney, Mike Burgess. Novel antagonists of TEAD palmitoylation inhibit the growth of Hippo-altered cancers in preclinical 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 3945.
Abstract 3949: Identification of a novel HASPIN inhibitor and the synthetic lethal partner by computational analysis
Authors: Kwon Eun-Ji , Mashelkar Karishma , Lee Hyuk Woo , Shin Yoon-Ze , Jeong Lak Shin , Cha Hyuk-Jin
Published: June 2022

HASPIN, a mitotic kinase to phosphorylate Histone H3 at centromere, has been studied as a promising target for anti-cancer therapy. However, as HASPIN belongs to atypical kinase, lacking the Asp-Phe-Gly (DFG) motif, development from the chemical library of kinase inhibitors with conventional pharmacophore, would be technically challenging. In particular, one of adenosine analogues (LJ4827), a potent inhibitor of multi-kinases, showing clear anti-cancer activity in vitro and in vivo, was predicted as a HASPIN inhibitor by computation analysis of its transcriptome profile in cancer cells to drug-omic data set in the connectivity MAP (cMAP) as it shared similar transcriptome profile of 5-iodotubercidin (5ITU). The specificity and potency as HASPIN inhibitor of LJ4827 (IC50 = 0.45 nM) validated by in vitro kinase screening and consequent 3D structure modeling. As expected, treatment of LJ4827 in cancer cell lines efficiently delayed mitotic progression without double strand break (DSB) unlike 5ITU and significantly attenuated Aurora B localization at centromere. Along with clinical significance of HASPIN expression in lung cancer patients, mitotic gene signature closely associated to high expression of HASPIN, revealed the poor prognosis. Additional computational analysis of kinase perturbation data to predict the dependency of mitotic kinase in the absence of HASPIN activity, revealed the synthetic lethal effect of cotreatment of the chemical inhibitor of BUB1, PLK1 or AURKA with LJ4827. These data suggest that combined inhibition of HASPIN with the novel inhibitor and key mitotic kinases for centromere/kinetochore regulation would be effectivity therapeutic approach for cancer therapy.

Citation Format: Eun-Ji Kwon, Karishma Mashelkar, Hyuk Woo Lee, Yoon-Ze Shin, Lak Shin Jeong, Hyuk-Jin Cha. Identification of a novel HASPIN inhibitor and the synthetic lethal partner by computational analysis [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 3949.
Abstract 3961: Cancer stem cells, not bulk tumor cells, predict mechanisms of resistance to SMO inhibitors in SHH medulloblastoma
Authors: George Joshy , Chen Yaohui , Abdelfattah Nourhan , Yamamoto Keiko , Adamson Scott , Choi Jong Min , Rybinski Brad , Srivastava Anuj , Kumar Parveen , Lee Min Gyu , Baskin David S. , Jiang Wen , Kim Betty Y. , Flavahan William , Chuang Jeffrey H. , Jung Sung Yun , Yun Kyuson
Published: June 2022

The emergence of primary and acquired treatment resistance significantly reduces the clinical utility of many effective targeted therapies. Both genetic and epigenetic mechanisms of drug resistance have been reported in literature; however, whether these mechanisms are stochastically selected in individual tumors or governed by a predictable underlying principle is unknown.Here, we report that one can predict a priori the resistance mechanism that will arise in individual SMO inhibitor (SMOi)-resistant SHH medulloblastoma (MB), based on different CSC phenotypes in each tumor. We show that the dependence of cancer stem cells (CSCs), not bulk tumor cells, on the targeted pathway (sonic hedgehog (SHH) pathway) determines the molecular mechanism of resistance in individual tumors. Using both spontaneous (Fsmo;GFAP-cre) and transplantable (Ptch+/-;p53) mouse models of SHH MB treated with a Smoothened inhibitor, sonidegib/LDE225, we show that genetic-based resistance occurs only when the CSCs depend on the targeted pathway. In contrast, SHH MBs containing SHH-dependent bulk tumor cells but SHH-independent CSCs (SI-CSCs), acquire resistance through epigenetic reprogramming. Mechanistically, we discovered that the elevated proteasome activity in SMOi-resistant SI-CSC MBs alters the tumor cell maturation trajectory through enhanced degradation of specific epigenetic regulators, including the histone acetylation machinery. Consequently, SMOi-resistant SI- SMOi-resistant SI-CSC exhibit a global reduction of H3K9Ac, H3K14Ac, H3K56Ac, H4K5Ac, and H4K8Ac marks and gene expression changes. These results provide new insights into how selective pressure on distinct tumor cell populations contributes to different mechanisms of resistance to targeted therapies and implicate histone acetylation in the process. This information can be clinically exploited to understand responses and resistance to SMOis and other targeted therapies.

Citation Format: Joshy George, Yaohui Chen, Nourhan Abdelfattah, Keiko Yamamoto, Scott Adamson, Jong Min Choi, Brad Rybinski, Anuj Srivastava, Parveen Kumar, Min Gyu Lee, David S. Baskin, Wen Jiang, Betty Y. Kim, William Flavahan, Jeffrey H. Chuang, Sung Yun Jung, Kyuson Yun. Cancer stem cells, not bulk tumor cells, predict mechanisms of resistance to SMO inhibitors in SHH medulloblastoma [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 3961.
Abstract 3962: Mechanisms of tumor recurrence and drug resistance in rhabdomyosarcoma
Authors: Moonamale Devmini C. , Wachtel Marco , Schäfer Beat W.
Published: June 2022

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma, which constitutes of two main histological subtypes. Alveolar Rhabdomyosarcoma (FP-RMS) is characterized by a fusion protein PAX3-FKHR, whereas embryonal Rhabdomyosarcoma (FN-RMS), which is the focus of this project, is more genetically heterogeneous. The prognosis for RMS has improved over the years, however the cure rates for recurrent or relapse disease remains dismal, warranting further identification of novel therapeutic interventions, which is the aim of this project. To this end, we performed two CRISPR knockout screens, using a kinome sgRNA library and a whole genome sgRNA library in combination with etoposide at a concentration of IC10, in order to identify genes that might confer resistance to chemotherapeutic drugs. In addition, the kinome screen was also designed to identify essential genes by using the treatment control samples as further time points. For treatment associated genes, we identified components of the JNK/P38 and Hippo signaling pathways among the top hits. We also found enrichment of a novel pathway involving HIFα stabilization via the genes LRRK2 and PTK6. Further and as expected, many DNA damage repair pathways were enriched. Finally in this category, players of the Wnt signaling pathway including JNK1, which is involved in non-canonical Wnt signaling were found to be enriched. As for essential gene identification, many cell cycle genes such as CDK6, PLK3 and PLK4 were among the top hits, as well as WEE1, which has been implicated in the context of RMS, were common between all the time points. Taken together, the CRISPR screens identified potential pathways that might be involved in resistance of FN-RMS cells towards etoposide. Selected candidates are being validated via an in vitro competition assay and drug screening is being performed to identify inhibitors that could be used to re-sensitize cells to treatment with standard chemotherapy.

Citation Format: Devmini C. Moonamale, Marco Wachtel, Beat W. Schäfer. Mechanisms of tumor recurrence and drug resistance in rhabdomyosarcoma [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 3962.