tbuckley/cabot-search huggingface dataset:
Abstract 1094: Utilizing CRISPR-Cas13 as a novel functional genomics tool to characterize PAX3-FOXO1 in alveolar rhabdomyosarcoma
Authors: Andrea Zhao, Lauren M. Brown, Mohamed Fareh, Paul G. Ekert
Journal: Cancer Research (2024)

Abstract Background: CRISPR-Cas13 (Cas13) is an RNA editing system that can knockdown gene transcripts with higher efficiency and specificity than RNA interference technologies. We are exploring the utility of Cas13 as a robust novel functional genomics tool to understand the biology of the PAX3-FOXO1(P3F1) gene fusion in alveolar rhabdomyosarcoma (ARMS). Our goal is to identify gene dependencies and novel druggable targets. [LB1] Aim: This study aims to functionally characterize P3F1 in paediatric cancer cell lines using Cas13. [LB2] Methods &amp; Results: Transient transfection and viral transduction of Cas13 vector systems were used to demonstrate that Cas13 knockdown of target gene expression can be achieved in ARMS cell lines. We developed guides to specifically target P3F1 expression and tested their efficiency in HEK293T cells. Cas13 function was validated with a mCherry knockdown proof-of-concept expression in the ARMS cell lines Rh5 and Rh30. 90-95% knockdown efficiency was seen over three independent experiments. Ten P3F1 breakpoint targeting guides were designed and screened first in a HEK293T P3F1 overexpression model to select optimal high efficiency targeting guides. The highest efficiency targeting guide showed a GFP reduction of 70% in three independent experiments. High-efficiency Cas13 guides were taken forward into Rh5 cells to knockdown endogenous P3F1 expression which were validated by western blotting and RT-PCR. Western blots of Cas13 sorted Rh5 cells showed that guide 1150 was able to knockdown P3F1 protein expression by ~70%. Discussion/Conclusion: These findings demonstrate the high efficiency and specificity of the Cas13 system to silence P3F1, and other oncogenic fusion drivers. Further studies will utilize Cas13 to characterize the molecular and cellular consequences of P3F1 gene-silencing in patient-derived ARMS cell lines. Citation Format: Andrea Zhao, Lauren M. Brown, Mohamed Fareh, Paul G. Ekert. Utilizing CRISPR-Cas13 as a novel functional genomics tool to characterize PAX3-FOXO1 in alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1094.
Abstract 6812: Durable control of brain tumors by glioma inhibitory macrophages and IL33
Authors: Shyam V. Menon, Xueqing Lun, Peipei Zeng, Jianbo Zhang, Bo Young Ahn, Henry Yu, Alisha Poole, Ngoc Ha Dang, Katalin Osz, Jennifer A. Chan, Daniela F. Quail, Stephen M. Robbins, Donna L. Senger
Journal: Cancer Research (2024)

Abstract Glioblastoma is the most common and deadly form of brain cancer. Even with aggressive treatment including surgery, chemotherapy, and radiotherapy, survival outcomes for newly diagnosed glioblastoma patients remains less than two years. Novel high throughput omics technologies have expanded our understanding of the role of innate immune system in brain tumors, that are generally believed to drive glioma progression and enable evasion of the adaptive immune system. However, targeting of this axis in the clinic remains an unmet opportunity. Previously, we discovered that the dual-function (secreted and nuclear) cytokine IL-33 is a crucial regulator of the inflammatory microenvironment that promotes glioma tumorigenesis through phenotypic and functional changes in the innate immune cell repertoire. Strikingly, when IL-33 is prevented from entering the nucleus, by deletion of its nuclear localization sequence (ΔNLS IL-33), but is still secreted, in vivo tumor growth is dramatically inhibited resulting in prolonged long-term survival. Using multiplex immunohistochemistry and spatial transcriptomics with temporal resolution across different stages of tumor progression, we identified a population of glioma-inhibitory macrophages (GIMs) unique to this suppressive environment. Assessment of GIMs in xenografts generated from patient brain tumor initiating cells found an enriched presence of these cells in xenografts with long-term survival (greater than 300 days) versus short-term survival (less than 100 days). The ability of GIMs to inhibit glioma progression was demonstrated when tumors established using a combination of ΔNLS IL-33 expressing cancer cells together with highly tumorigenic cells resulted in a growth inhibitory environment that significantly extended survival. A deeper molecular characterization of this phenotype and development of clinical strategies are currently underway. Citation Format: Shyam V. Menon, Xueqing Lun, Peipei Zeng, Jianbo Zhang, Bo Young Ahn, Henry Yu, Alisha Poole, Ngoc Ha Dang, Katalin Osz, Jennifer A. Chan, Daniela F. Quail, Stephen M. Robbins, Donna L. Senger. Durable control of brain tumors by glioma inhibitory macrophages and IL33 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6812.
Abstract 1059: Biomarkers analysis on samples from patients in EVT801 clinical trial: Patient characterization and immunomonitoring
Authors: Lise Davenne, Michael Fitzgerald, Pierre-Benoit Ancey, Oona Delpuech, Céline Poussereau-Pomié, Michael Esquerre, Michael R. Paillasse, Marie Mandron, Philippe Rochaix, Maha Ayyoub, Clara Maria Scarlata, Christine Caux, Christophe Caux, Philippe Cassier, Carlos Gomez-Roca, Jean-Pierre Delord, John Friend, Pierre Fons
Journal: Cancer Research (2024)

Abstract Introduction: EVT801 is a highly selective small molecule inhibitor of VEGFR3 that acts by inducing tumor (lymph)-angiogenesis normalization in and around the tumor. EVT801 has shown compelling activity in a wide range of cancer models showing a decrease of tumor hypoxia and an increase of CD8pos T-cells infiltration into the tumor micro-environment. EVT801 was well-tolerated in preclinical toxicology studies. A phase I study is underway focusing primarily on understanding the safety, tolerability, and pharmacokinetics of EVT801 across a range of doses (NCT05114668). Methods: The first stage of the phase I trial aims to determine the maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) for EVT801. Clinical samples from enrolled patients have been collected to explore preliminary signals of the biological activity of the drug and characterize tumor phenotypes. Patient characterization includes histology and mRNA signature in archival biopsies. Target engagement and pharmacodynamics effects are investigated by immunomonitoring as well as assessment of a defined set of proteins as markers of inflammation and angiogenesis as identified in preclinical in vivo models. Conclusion: Our first biomarkers analyses on a subpopulation of patients with High Grade Serous Ovarian Cancer (HGS-OC) enrolled in the first stage of the study showed that levels of VEGFR3 expression had a negative correlation with CD8 positive T-cells infiltration in the tumor microenvironment and a tendency to positive correlation with a PD1 mAb resistance signature. VEGFR3 expression also tends to be correlated with higher levels of hypoxia (CAIX labelling). Our working hypothesis is that patients with hypoxic HGS-OC tumors with high VEGFR3 expression may benefit from EVT801 treatment; this will need to be reinforced by inclusion of additional patients in dedicated PD biomarkers cohorts during stage 2 of the clinical trial. Citation Format: Lise Davenne, Michael Fitzgerald, Pierre-Benoit Ancey, Oona Delpuech, Céline Poussereau-Pomié, Michael Esquerre, Michael R. Paillasse, Marie Mandron, Philippe Rochaix, Maha Ayyoub, Clara Maria Scarlata, Christine Caux, Christophe Caux, Philippe Cassier, Carlos Gomez-Roca, Jean-Pierre Delord, John Friend, Pierre Fons. Biomarkers analysis on samples from patients in EVT801 clinical trial: Patient characterization and immunomonitoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1059.
Abstract 6583: Developing a selective cancer chemotherapy by small molecule-based targeting of PCNA protein
Authors: Jennifer Jossart, Long Gu, Pouya Haratipour, Caroline Li, Robert Lingeman, Robert Hickey, Linda Malkas, John J. Perry
Journal: Cancer Research (2024)

Abstract Proliferating cell nuclear antigen (PCNA) is a proliferation biomarker in a variety of human tumors and has critical cellular functions in DNA replication and repair. We previously identified a unique cancer-associated isoform of the protein, termed caPCNA, which allows for structure-based drug discovery studies to develop inhibitors for selective therapeutic targeting of cancer cells. Distinct strategies have been used to develop caPCNA targeting agents, which includes peptide and small molecule-based compounds, although the success in developing therapeutically tractable compounds has been limited so far. Here, we present the crystal structures and corresponding biochemical characterizations of our novel small molecule-based caPCNA inhibitors, which bind into the PIP-box binding pocket of PCNA. Our lead compound selectively kills cancer cells, and it appears to induce replication stress, apoptosis and increase cancer cell sensitivity to genotoxic agents, while these effects are not observed in non-malignant cell controls. This caPCNA inhibitor is orally administrable, metabolic stable and it suppresses tumor growth as a monotherapy or as a combination treatment and is now an investigational new drug in Phase 1 clinical trials. PCNA plays a critical role enabling the replication fork to proceed from transcription replication conflict sites, by temporarily dislodging RNA polymerase II. Thus, structure-based drug discovery against caPCNA may open a novel therapeutic avenue for the development of selective chemotherapeutics, through targeting a cancer cell’s vulnerability to unresolved transcription replication conflicts. Citation Format: Jennifer Jossart, Long Gu, Pouya Haratipour, Caroline Li, Robert Lingeman, Robert Hickey, Linda Malkas, John J. Perry. Developing a selective cancer chemotherapy by small molecule-based targeting of PCNA protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6583.
Abstract 7044: Transcriptome and TCR repertoire in human telomerase reverse transcriptase peptide vaccine treated metastatic prostate cancer patients
Authors: Reetta Nätkin, Julia Elisabeth Simensen, Nikolai Engedal, Espen Basmo Ellingsen, Marita Westhrin, Ingunn Westgaard, Eivind Hovig, Wolfgang Lilleby, Matti Nykter, Alfonso Urbanucci
Journal: Cancer Research (2024)

Abstract Human telomerase reverse transcriptase (hTERT) vaccine has found to be effective for most of the participated de novo metastatic prostate cancer patients in clinical studies. Nevertheless, the molecular mechanisms behind the immune response induced by the hTERT vaccine are not fully elucidated. Among the cohort of 21 patients treated with androgen deprivation therapy, radiotherapy and hTERT vaccine, we used RNA-sequencing and TCR-sequencing (20 and 14 pre-treatment and 20 and 21 post-treatment samples, respectively) to inspect the changes in transcriptome and TCR-repertoire after the hTERT vaccine treatment and differences between patients that had early response and patients that had late or no response. We found multiple hallmarks being shifted after the treatment and being different between early and late responders. Also, a change in activity of multiple gene set signatures was detected after the treatment and activity of part of the gene set signatures predicted survival. Deconvolution of cell type composition from RNA-sequencing data revealed changes in immune cell abundances after the treatment. TCR-sequencing revealed changes in composition and diversity of TCR repertoires after the treatment and these differed between early and late responders. The results of this study will help to predict patients’ responses to the cancer vaccine and to further understand the mechanisms behind successful cancer vaccines. Citation Format: Reetta Nätkin, Julia Elisabeth Simensen, Nikolai Engedal, Espen Basmo Ellingsen, Marita Westhrin, Ingunn Westgaard, Eivind Hovig, Wolfgang Lilleby, Matti Nykter, Alfonso Urbanucci. Transcriptome and TCR repertoire in human telomerase reverse transcriptase peptide vaccine treated metastatic prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7044.
Abstract 1522: Tumor-associated fibrosis leads to an increase in T-cell exhaustion and impairs tumor control in non-small cell lung cancer
Authors: Usman Y. Panni, Varun Shenoy, Brett Knolhoff, Faiz Ahmed, Brett Herzog, William G. Hawkins, John A. Olson, David G. DeNardo
Journal: Cancer Research (2024)

Abstract Lung cancer is the leading cause of cancer-related mortality with non-small cell lung cancer (NSCLC) being the most predominant type. Recent studies have shown the efficacy of anti-PD1 inhibitors which improved overall survival. However, this efficacy is limited to a subset of patients due to the intrinsic tumor resistance to immune checkpoint inhibitors. Our group recently demonstrated that tumor-associated fibrosis negatively correlates with T cell-mediated immune response in lung cancer. This is largely dependent on Col13a1-expressing cancer-associated fibroblasts (CAFs), which result in recruiting the immunosuppressive macrophages and regulatory T cells while limiting dendritic cells and CD8 T cell infiltration. Here, we characterize the effect of fibrosis on the TME including TILs and macrophages using an orthotopic KRAS and P53-driven murine NSCLC model (KPL86-mCher model) with FITC-induced fibrosis.Fibrosis significantly increased the number of cancer-associated fibroblasts and collagen density in KPL86-mCher lung tumors. Following carboplatin + pemetrexed and Anti-PD1 treatment, we observed no increase in tumor-infiltrating CD4+ and CD8+ T cells or decrease in tumor burden when compared with the non-fibrotic tumors. Rather, we observed a significant increase in exhausted T cells marked by high Tim3, PD1, and TOX in fibrotic KPL86-mCher tumors. Corresponding to these impaired T-cell responses, we found an increase in tumor-infiltrating alveolar macrophages with immunosuppressive phenotype under fibrotic tumor conditions. Our data suggests that tumor-associated fibrosis leads to an increase in CD8+ T cell exhaustion as well as immunosuppressive macrophage infiltration in our spontaneously derived murine model on NSCLC. Overall, this indicates that targeting tumor fibrosis may significantly improve the efficacy of immunotherapy which would have translational implications for patients with immunotherapy-resistant NSCLC. Citation Format: Usman Y. Panni, Varun Shenoy, Brett Knolhoff, Faiz Ahmed, Brett Herzog, William G. Hawkins, John A. Olson, David G. DeNardo. Tumor-associated fibrosis leads to an increase in T-cell exhaustion and impairs tumor control in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1522.
Abstract 7022: Whole genome and reduced representation enzymatic methyl-seq enable cost effective methylomes
Authors: Vaishnavi Panchapakesa, Chaithanya Ponnaluri, Daniel Evanich, Ariel Erijman, Bradley Langhorst, Louise Williams
Journal: Cancer Research (2024)

Abstract DNA methylation is an epigenetic regulator of gene expression with important functions in development and diseases such as cancer. Traditionally, sodium bisulfite conversion was used to distinguish 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC) from cytosines. However, this method damages DNA and introduces significant sequencing bias. NEBNext® Enzymatic Methyl-seq (EM-seq™) is an enzymatic conversion approach that minimizes DNA damage therefore enabling longer insert sizes, lower duplication rates and a more accurate quantification of methylation in DNA samples with inputs ranging from 10 ng to 200 ng. An improved enzymatic conversion method, EM-seq v2, has a more streamlined workflow and an expanded DNA input range of 100 pg to 200 ng. Libraries made using NA12878 genomic DNA show improved yields, consistent global methylation and an even GC bias representation. The increased library yields subsequently reduces sequencing duplications and costs. Sequencing costs are an important consideration when performing whole genome methylome analysis as this often needs high sequencing depth to attain meaningful coverage to accurately call methylation. Reduced representation libraries, a commonly used method to investigate DNA methylation, relies on enrichment of CpGs within libraries digested using MspI. These CpGs are commonly found in regulatory regions. Although lacking comprehensive CpG coverage, for many researchers this method provides enough cost effective CpG methylation coverage for their studies. Reduced Representation EM-seq libraries (RREM-seq) and Reduced Representation Bisulfite libraries (RRBS) were made using 200 ng to 100 pg of NA12878 DNA. EM-seq adaptors were ligated to MspI digested DNA followed by conversion using either EM-seq v2 or sodium bisulfite. Libraries were PCR amplified and sequenced on an Illumina NovaSeq platform. RREM-seq resulted in higher yields and reproducible results regardless of input amounts compared to RRBS libraries. RREM-seq libraries also identified a higher number of CpGs with even coverage facilitating accurate methylation calling. RREM-seq libraries have superior sequencing metrics resulting in robust methylation profiling. Focusing on a subset of the genome generates higher coverage DNA methylation data at a lower DNA sequencing cost. Citation Format: Vaishnavi Panchapakesa, Chaithanya Ponnaluri, Daniel Evanich, Ariel Erijman, Bradley Langhorst, Louise Williams. Whole genome and reduced representation enzymatic methyl-seq enable cost effective methylomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7022.
Abstract 1411: Cell-type-specific transcriptomic immune aging clocks reveal clinically relevant associations with chronic illnesses including cancer
Authors: Yael Gurevich-Schmidt, Kun Wang, Di Wu, Sanna Madan, Vishaka Gopalan, Sanju Sinha, Binbin Wang, Saugato Rahman Dhruba, Alejandro A. Schäffer, Eytan Ruppin
Journal: Cancer Research (2024)

Abstract The aging of the immune system has profound implications for individual immune responses, yet precise quantification of immune age remains a challenge. Analyzing single-cell peripheral blood mononuclear cells (PBMC) transcriptomics data from 981 healthy individuals, we developed IMMClock (IMMune Clock), a human immune age clock derived from single cell transcriptomics. IMMClock is the first to accurately assess the age of three major immune cell types, CD8+ T cells, CD4+ T cells and NK cells, at the individual level. Testing the ability of these clocks to capture the dynamic aging process, we show that they reliably predict individuals' chronological ages across seven publicly available single-cell transcriptomics datasets and the large Framingham bulk transcriptomics cohort. IMMClock identifies cell type specific age-related pathways including apoptosis, interferon gamma response, and cytokine response. The application of IMMClock reveals several associations between immune aging of different cell-types and key clinical phenotypes during aging: it recapitulates the established observation of higher immune age in males compared to females and uncovers higher immune age in CD8+ T cells of smokers and individuals with chronic illnesses including cancer. Analyzing cancer patients’ data, IMMClock readings strongly correlate with CD8+ T cell exhaustion levels, and, quite strikingly, highlights an elevated immune age in the tumor microenvironment compared to blood. Citation Format: Yael Gurevich-Schmidt, Kun Wang, Di Wu, Sanna Madan, Vishaka Gopalan, Sanju Sinha, Binbin Wang, Saugato Rahman Dhruba, Alejandro A. Schäffer, Eytan Ruppin. Cell-type-specific transcriptomic immune aging clocks reveal clinically relevant associations with chronic illnesses including cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1411.
Abstract 3791: A prognostic signature based on inherited genetic polymorphisms for patients with uveal melanoma
Authors: Thibault Verrier, Anaïs Le Ven, Alexandre Houy, Agathe Garcia, Pierron Gaelle, Nathalie Cassoux, Manuel Rodrigues, Josselin Noirel, Marc-Henri Stern
Journal: Cancer Research (2024)

Abstract Introduction: Uveal melanoma (UM) is the most frequent eye malignancy in adults. Two UM subtypes exist, defined by Gene Expression Profiling or by genomic status: GEP class 1/disomy 3 [D3] and GEP class 2/monosomy 3 [M3] defining low-risk or high-risk of developing metastasis, respectively. However, these prognostic signatures require tumor samples, which may be difficult to obtain. We previously identified by a genome-wide association study (GWAS) frequent single nucleotide polymorphisms (SNP) in IRF4 and HERC2 associated with risk for D3 or M3 UM, respectively (1). Here, we aim to design a prognostic signature for the D3 and M3 UM subtypes based on these constitutive polymorphisms, available from blood samples. Experimental Procedures: We used the GWAS cohort of 1476 UM patients genotyped on GSA or Omni5. Risk SNPs coupled with clinical variables were used on a cohort of 497 UM patients (191 D3, 306 M3) in a statistical machine learning process to define a predictive signature of UM subtype. A cohort of 852 UM patients was used for validation. Results: We retained two SNPs, rs12203592 (in IRF4) and rs12913832 (in HERC2), tumor diameter and thickness, age at diagnosis and gender. We validated the predictive model in the validation cohort. The predicted high-risk group showed a decrease in time to relapse (TR) (HR: 3.72 [2.57 - 5.41], logrank p-value = 1.35E-15), overall survival (OS) (HR: 3.85 [2.58 - 5.73], logrank p-value = 1.24E-14) and progression-free-survival (PFS) (HR: 3.61 [2.32 - 5.61], logrank p-value = 6.55E-11), compared with the predicted low-risk group. Conclusion: Common germline polymorphisms contribute to the prediction of metastatic risk in patients with localized uveal melanoma. This information could be especially useful when access to the tumor is problematic. 1. Mobuchon et al, J Natl Cancer Inst, 114, 2, (2022) Citation Format: Thibault Verrier, Anaïs Le Ven, Alexandre Houy, Agathe Garcia, Pierron Gaelle, Nathalie Cassoux, Manuel Rodrigues, Josselin Noirel, Marc-Henri Stern. A prognostic signature based on inherited genetic polymorphisms for patients with uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3791.