Expression Profiling Of Tumors Research Articles

RNA-Seq based gene expression profiling of baseline and on-treatment breast tumors to predict response to HER2-directed therapy, without chemotherapy (TBCRC026).

530 Background: Robust biomarkers are needed to personalize treatment in early stage HER2-positive breast cancer. The TBCRC026 trial (n=83) demonstrated that early metabolic changes on FDG-PET/CT [>40% decline in SULmax at day 15 post treatment initiation (C1D15); C1D15 SULmax] were associated with response to neoadjuvant trastuzumab/pertuzumab (HP), without chemotherapy, in an ER-negative, HER2-positive cohort (NCT01937117). We investigated the association of RNA sequencing (RNAseq)-based gene expression signatures (GES) at baseline and on-treatment (C1D15), with early metabolic change on PET/CT and clinical outcomes. Methods: RNAseq (664 GES) was performed on baseline (n=56) and C1D15 (n=31) tumor biopsies (25 paired samples available). Association with pCR following HP (secondary), PET/CT parameters and survival (exploratory) was studied. Gene expression changes between baseline and C1D15 samples were tested by Wilcoxon signed-rank test. Association between GES and PET/CT parameters and GES with pCR were evaluated using logistic regression models. Cox regressions were performed to study the association between PET/CT SULmax at C1D15 and a B cell signature and relapse-free survival (RFS). The likelihood ratio test was used to compare the prognostic ability of two nested Cox models. Results: Both RNAseq and clinical outcomes were available in 53/83 patients. HER2-enriched (HER2-E) intrinsic subtype comprised 71% (40/56) of cases at baseline and 61% (19/31) at D15; basal subtype for 27% (15/56) and 39% (12/31), respectively. pCR rate was 19% (10/53) with all but one exhibiting HER2-E subtype. One basal-like sample underwent pCR, with downregulation of HER2 amplicon and IgG signatures slightly more prominent. Gene expression changes from baseline to C1D15 were analyzed; ERBB2was downregulated after HP (p = 0.007), B cell signatures, CD4, CD8, natural killer cells and Treg cells were significantly upregulated at C1D15. GES associated with 40% decline in SULmax at C1D15 included ERBB2 amplicon genes ( ERBB2, GRB7) and correlation to the HER2-E subtype, whereas basal-like signatures were significantly associated with lack of response on PET at C1D15. Univariate logistic regression analysis suggested that FOS-JUN, fibroblast and B cell GES at baseline were significantly associated with pCR (p=<0.05). Finally, the addition of a B cell signature improved the prognostic value of C1D15 SULmax on PET/CT for RFS (p < 0.05). Conclusions: In this unique cohort with ER-negative, HER2-positive breast cancer treated with neoadjuvant HP without chemotherapy, HER2-E tumors and those with increased immune signaling were more likely to respond to HER2-directed therapy. Combining C1D15 SULmax on PET/CT and a B cell signature provided improved prognostic information and should be further explored as candidate biomarkers in this setting. Clinical trial information: NCT01937117 .

Abstract 3793: Mutational and gene expression profiles of the primary tumor and matched brain metastasis of epithelial ovarian cancer reveal differences in LAMC3 and MYC target genes

Abstract Introduction: Brain metastases (BM) from Epithelial ovarian cancer (EOC) are a rare and late-stage disease manifestation, very poorly biologically characterized. We performed a multi-omics characterisation of primary EOC samples (PT) and the matched BM, to assess differences in mutational and gene expression profiles. Material and Methods: We retrospectively collected data from EOC patients treated at Fondazione Policlinico Gemelli IRCCS who underwent surgical removal of BM between 2011 and 2022. We collected the formalin-fixed, paraffin-embedded (FFPE) tissue of the PT and the matched BM. An evaluation of single nucleotide variants (SNVs), insertions/deletions (indels) and copy number variations (CNVs) in 523 genes as well as known and unknown fusions and splicing variants in 55 genes was performed (Illumina TruSight Oncology 500 high throughput). Moreover, a bulk RNAseq analysis was conducted utilizing the NEGEDIA Digital mRNA-seq clinical-grade sequencing service. We acknowledged as differentially expressed genes if they exhibited a −1≤log2 (Fold Change)≥1 and pAdj≤0.05. To ensure specificity in identifying differential gene expressions pertinent to our tumoral tissues, genes associated with brain-related functionalities were systematically excluded from downstream analyses and identified through meticulous screening via the Human Protein Atlas. Clonal evolution was evaluated with two distinct algorithms: Pyclone VI and Sciclone. Results: Matched PT/BM FFPE samples were collected from 10 different patients. We identified 21 SNVs: most of them were shared between the PT and the matched BM and TP53, CCND1, IRS2, and FGFR4 mutations emerged as the most frequently detected (shared in 70%, 50%, 50%, and 40% of the cases, respectively). Among 56 CNV detected, most of them were exclusive of the BM samples. Specifically, MYC family genes (MYC, MYCL, MYCN) were the most frequently amplified. At the clonal analysis, each patient showed at least two clusters. The majority of clusters turned out to be consistent among PT and BM demonstrating monoclonal evolution. Gene set variation analysis discovered 50 different gene sets between the PT and the BM (p < 0.05). Among them, the E2F targets, G2M checkpoint, MYC targets V1 and MYC targets v2 hallmarks showed higher expression in the BM. We identified 110 significant differentially expressed genes between PT and BM samples, including 27 upregulated and 83 downregulated genes. The gene LAMC3 emerged as notably upregulated in metastatic contexts. Conclusion: This is the largest cohort present in the Literature of EOC PT and matched BM characterized with a multi-omics analysis. The intersection of LAMC3 and MYC target genes within the MET signalling network, a pathway recognized for bolstering cell motility, introduces a compelling avenue for future research. Citation Format: Rita Trozzi, Camilla Nero, Marianna Buttarelli, Marcello Salvi, Luciano Giacò, Angelo Minucci, Claudia Marchetti, Carolina Sassu, Federica Persiani, Lorenzo De Marco, Simona Duranti, Camarda Floriana, Valentina Iacobelli, Ilenia Marino, Luca Mastrantoni, Flavia Giacomini, Alessia Piermattei, Mariagrazia Valentini, Tina Pasciuto, De Bonis Maria, Giuseppina Raspaglio, Diana Giannarelli, Anna Fagotti, Davide Cacchiarelli, Giovanni Scambia. Mutational and gene expression profiles of the primary tumor and matched brain metastasis of epithelial ovarian cancer reveal differences in LAMC3 and MYC target genes [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 3793.

Abstract 6489: Paradoxical activation of kinases occurs directly with ATP-competitive kinase inhibitors and is observable biochemically at physiologically relevant drug concentrations

Abstract We sought to screen all FDA approved, cancer-indicated, kinase targeting drugs using the gold standard radiometric biochemical activity assay (HotSpot) against the largest collection of kinases in the world (>100 drugs against 735 total kinases including 395 wild type and 340 mutants). One of these drugs, mitapivat, is an activator of Pyruvate Kinase. Here, we show mitapivat-driven biochemical inhibition of several, but not all, mutants of c-KIT. We also observed the equal and opposite phenomenon whereby many ATP-competitive kinase inhibitors demonstrate biochemical activation of certain kinases, several of which seem particularly susceptible to the effect. Previous work by other investigators (at Genentech and the Rosen lab, MSKCC) demonstrated that PLX4720 (precursor to Vemurafenib) paradoxically activated wild type BRAF, but not BRAF mutants, by driving dimerization of the kinase in drug concentrations insufficient to inhibit both members of the dimerized pair. Our data suggests this phenomenon may be more widespread than previously appreciated and fully observable in vitro. Such paradoxical activation could be driving both toxicities and inefficacy against cancers expressing some of these activatable kinases. This information could suggest novel opportunities for combination therapy and improve outcomes by contraindicating certain medication from use based on tumor expression profile. Citation Format: Christian M. Loch, Mia Eason, Shuguang Liang, Alison Goupil, Anthony Acinapura, Jamie Purcell, Maia Domiguez. Paradoxical activation of kinases occurs directly with ATP-competitive kinase inhibitors and is observable biochemically at physiologically relevant drug concentrations [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 6489.

Abstract 5494: Spatial organization between claudin and the tumor microenvironment in head and neck squamous cell carcinomas

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth leading form of cancer worldwide and one of the most difficult and challenging malignancies, with a high level of heterogeneity and a wide range of treatment responses. Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer and accounts for over 90% of cancers that develop within the mucosal epithelium of the oral cavity. Additional carcinogenic risk factors, including tobacco, alcohol and HPV infection are also associated with the pathogenesis of oral carcinogenesis. Unfortunately, today the failure of standard treatment modalities, such as surgery, radiotherapy, and chemotherapy underscore the need to identify better biomarkers of this disease. Advances, especially in spatial technology have created unprecedented opportunities to identify cell types and biomarkers of disease processes, revealing important relationships within the tumor microenvironment (TME) that allow a detailed characterization of specific cell phenotypes defined by co- or lack of expression of multiple markers that may help in predicting clinical responses and mechanisms of resistance to therapy. Recent findings indicate that abnormal expression of claudins (CLDNs) is closely related to the occurrence, progression and prognosis of many solid tumors. Specifically, CLDN6 is expressed in numerous tumors but rarely found in adult normal tissues making it a potential therapeutic target in head and neck tumor types. To investigate this further, expression of CLDN6 and the its relationship with changes within the TME of cohort OSCC tumors was determined using a proprietary comprehensive spatial workflow and gene expression analysis. Methods: A retrospective analysis was performed on biopsied Formalin-Fixed Paraffin-Embedded (FFPE) samples from OSCC patients with and without a history of smoking and treated +/- chemotherapy. Patient samples representing matched normal tissue to serve as controls were compared to samples derived from OSCC tumors. In order to perform a comprehensive expression profiling of the TME from OSCC tumors we used the spatial MultiOmyx™ immunofluorescence (IF) multiplexing assay with transcriptomic information from bulk mRNA gene expression acquired using the NanoString nCounter® PanCancer IO 360™ panel that includes the Tumor Inflammation Signature (TIS), as well as targets that characterize immune hot, desert, and cold regions in the stroma and tumor epithelium. Results: We characterized the expression of CLDN6 across the different cohorts of OSCC samples and further investigated the relationship within the tumor immune microenvironment coupled to genes related to the prognosis of OSCC. A better understanding of the TME and gene expression profiling of OSCC tumors and the effect of smoking will allow us to tailor the treatment of patients to continue to improve outcomes. Citation Format: Kirsteen Maclean, Lakshmi Chandramohan, Maryam Rohafza, Harry Nunns, Erinn Parnell, Qingyan Au, Anna Juncker-Jensen. Spatial organization between claudin and the tumor microenvironment in head and neck squamous cell carcinomas [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 5494.

Abstract 1040: Development of a novel RNA-based microsatellite stable predictive response signature (MSS-PRS) to identify MSS colorectal cancer (CRC) patients with a microsatellite instability-high (MSI-H) molecular phenotype

Abstract Introduction: MSI-H and mismatch repair deficient (dMMR) tumors are associated with favorable immune checkpoint inhibitor (ICI) responses (PMID: 3326454). However, up to 95% of CRC tumors are MSS/proficient MMR (pMMR) leading to poorer prognosis and treatment outcomes than MSI-H/dMMR patients (PMID: 12867608, 26028255). In MSS/pMMR metastatic CRC, multiple combination therapies are being investigated despite a lack of biomarkers to guide therapy. Therefore, we developed an RNA-based MSS-PRS with a primary objective to identify MSS tumors that have MSI-H/dMMR molecular features indicative of ICI response. Experimental Procedures: The MSS-PRS was developed using the TCGA CRC cohort (COAD; n=268). Training labels were assigned using the MSI Mantis score and mutation status from 8 genes with reported MSI association. Samples called “activated” had both a gene mutation and an MSI Mantis score > 0.4 (i.e., MSI-H), and samples called “non-activated” were wild type for all genes and had MSI Mantis score < 0.4. All other samples were designated “ambiguous” and excluded from training. Two thirds of the non-ambiguous samples were assigned to the classifier training set and all remaining samples were assigned to the test set. Using ClaNC software (PMID: 16269418) and cross-validation in the training set, a nearest centroid classifier was developed from a set of high mean, high variance candidate genes to select an optimal gene set to separate activated and non-activated groups. The classifier performance was evaluated in the test set. Results: The MSS-PRS contained 112-genes enriched for mismatched DNA binding, DNA damage repair, PD-L1, innate and adaptive immune response, cellular immunity, and cytokines. In the test set, the classifier called 44 of 46 MSI-H samples activated; of the remaining 77 samples that were MSS tumors, 38 were called activated and 39 were called not activated. Visual inspection of heat maps of tumor by MSI/MSS-PRS status and their association with TMB and CRC-related mutations suggest greater differences between MSS tumors called activated and MSI-H tumors than between MSS tumors called activated and MSS tumors called not activated. In contrast, immune marker expression profiles in MSS tumors called activated were markedly more similar to MSI-H tumors compared to MSS tumors called non-activated. Summary and Conclusions: Herein we described the development of a novel MSS-PRS that captures an MSI-H/dMMR-like molecular phenotype in a subset of patients with MSS tumors despite their lack of high TMB or overt MSI defects. Based upon these initial findings, further development of the MSS-PRS and its clinical validation as a tool to select patients with MSS tumors who may benefit from ICI-containing treatment regimens is warranted. Citation Format: Kirk Pappan, Gregory Mayhew, Jonathan Shepherd, Yuelong Guo, Kirk Beebe, Joel Eisner, Michael Milburn. Development of a novel RNA-based microsatellite stable predictive response signature (MSS-PRS) to identify MSS colorectal cancer (CRC) patients with a microsatellite instability-high (MSI-H) molecular phenotype [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 1040.

Abstract 2268: CREDAC: Copy number-based reference-free expression deconvolution analysis of cancers

Abstract Introduction: Analyses of bulk RNA sequencing data are central to most large-scale tumor sequencing studies. Bulk expression data represent population averages and its interpretation is confounded by both normal cell contamination and somatic copy number alterations. Several computational methods to deconvolve have been developed recently. However, these methods often rely on a set of cell-type-specific reference signatures and ignore the effect of copy number changes. Methods: To address these issues, we have developed a method that formalizes the relationship between allele-specific copy number, expression and sample purity to deconvolve the expression profiles of tumor and normal cells from bulk RNA-seq in an unbiased manner. Our method was applied to sequencing data produced by the TRACERx consortium, a longitudinal study with multi-region whole-exome and RNA-seq of non-small-cell lung cancers. A total of 414 primary tumor regions and 140 adjacent normal tissue samples from 140 patients with matched DNA and RNA-seq data were processed. Results: We were able to directly deconvolve a median of ~2,000 genes per sample and indirectly infer tumor and normal expression profiles of ~10,000 genes using an scaled and weighted mean approach. The accuracy of the deconvolution was validated using (1) in-silico mixtures of patient-derived tumor and normal cells, (2) pseudo-bulk scRNAseq and (3) in regions with loss of heterogeneity (LOH) directly on the bulk sequencing data, where the total fraction of expression attributed to tumor cells can be computed directly using somatic mutations. CREDAC allows for single sample bulk-level tumor-normal differential expression analysis and revealed a strong and constitutive genome-wide overexpression in cancer compared to admixed normal cells, with a more pronounced overexpression in lung squamous cell carcinoma than lung adenocarcinoma (p<0.001). CREDAC adjusts for variations in copy number, thus facilitating the investigation of cancer-specific dosage compensatory responses triggered by aneuploidy. We revealed that a majority of genes (~65%) exhibit proportionality to copy number alterations, while a substantial proportion (~35%) shows anti-scaling or dosage compensation strategies. We show an enrichment of genes involved in cell cycle and expression processes within the dosage compensated group and revealed methylation as a key mechanism driving this compensatory process. Conclusion: Overall, our results suggest that CREDAC is able to accurately disentangle the expression of tumor and normal cells from bulk RNA-seq without any previous knowledge. It has potential applications in many studies that include matched RNA-seq and copy number data and can provide new insights functional characterization, the taxonomy of cancer, and tumor evolution. Citation Format: Carla Castignani, Jonas Demeulemeester, Oriol Pich, Tom Lesluyes, Robert E. Hynds, David R. Pearce, Elizabeth Larose Cadieux, Stefan C. Dentro, TRACERx Consortium, Nnenna Kanu, Charles Swanton, Peter Van Loo. CREDAC: Copy number-based reference-free expression deconvolution analysis of cancers [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 2268.

Abstract 5496: Multi-omic spatial analysis of the tumor microenvironment in gliomas

Abstract Introduction: The diverse tumor environment of high-grade glioma, which remains refractory to treatment, demands new innovative, multi-omic approaches to characterize tumor heterogeneity and expression profiles associated with progression and therapeutic response. Approach: We have applied an integrated, multi-omic approach using paired spatial in situ sequencing (Xenium) and protein profiling (PhenoCycler® Fusion) of glioma tissue to identify distinct glioma cellular phenotypes, activation states and metabolic pathways at true single cell resolution. Our multi-omic approach used hundreds of phenotypic RNA target probes and >50 antibodies in an unbiased single-cell analysis of primary, recurrent, and IDH1 mutant/wild type tumors with a range of heterogeneity. Summary: We were able to distinguish differentially regulated genes and pathways between aggregated primary and recurrent GBM, including cell cycle pathways being upregulated in primary vs recurrent GBM glial cell clusters and a down regulation of ERBB4 signaling in vascular cell clusters in primary GBMs. Our data also suggests that SOX11, known to be involved in tumorigenesis, is >2-fold upregulated in annotated tumor cells in primary compared to recurrent tumors. We were also able to quantitatively localize the expression of Epidermal Growth Factor Receptor, variant III (EGFRvIII) to specific tumor-cell sub-types, which is important given its association with therapeutic resistance. Further, we have been able to establish the cellular neighborhoods and cell-cell and receptor-ligand relationships within the tumors. Our spatial protein analysis identified distinct tumor and immune phenotypes with varying abundance of myeloid and lymphoid populations in IDH1 wt and mt tumors. Moreover, spatial proximity analyses and cellular neighborhood analyses revealed differences in higher order organizational landscapes that may contribute to the differential outcomes across wt and mt tumor subtypes. Conclusion: Multiomic spatial analysis enables deeper characterization of the glioma cellular and functional landscape to broaden our understanding of the key TME features that contribute to disease pathogenesis and prognoses. Our study provides an analytical framework to combine RNA and protein-based spatial data for a holistic investigation into a variety of glioma subtypes and aid in the identification of novel biomarkers, spatial neighborhoods, and functional states that drive glioma progression. Citation Format: Dmytro Klymyshyn, Vaibhav Jain, Lauren Whaley, Emily Hocke, Bassem Ben Cheikh, Alan Smith, Karen Abramson, Nadine Nelson, Diane Satterfield, Elizabeth Thomas, Giselle Lopez, Seetha Hariharan, Michael Brown, Niyati Jhaveri, Roger McLendon, David Ashley, Matthew Waitkus, Simon Gregory. Multi-omic spatial analysis of the tumor microenvironment in gliomas [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 5496.

Abstract 5301: Tumor-targeted activation of CD137 using Bicycles: New insights into mechanism of action and discovery of BT7455, a clinical candidate for the treatment of EphA2-expressing cancers

Abstract Introduction: Clinical studies in cancer patients have validated CD137 agonism as an activator of the immune system to enable tumor rejection. We have demonstrated that small, chemically synthetic bicyclic peptides can drive tumor-localized agonism of CD137 and anti-tumor immunity in mouse models. Here, we report the next stage of our work - delving into the mechanism of action of these novel agents and extending our program to serve patients whose tumors express EphA2. Experimental Procedures: MultiOmyx™ hyperplexed immunofluorescence assay was used to evaluate the expression of CD137 and EphA2 in head and neck squamous cell carcinoma samples. Human PBMC/tumor cell co-culture assays were used to assess CD137 Bicycle TICA™ in vitro bioactivity and syngeneic mouse tumor models were used to evaluate CD137 Bicycle TICA anti-tumor activity as mono- or combination therapies. Pharmacodynamic activity was evaluated by transcriptional profiling using NanoString assays or single cell RNA sequencing. Summary of the Data: Studies utilizing a syngeneic mouse model and deep RNA sequencing of tumors from CD137 Bicycle TICA-treated mice implicated intratumoral dendritic cells in addition to T cells as early responders to localized CD137 agonism and potential contributors to the anti-tumor response. Furthermore, a mouse efficacy model demonstrated synergy with checkpoint inhibitor therapy. Multiplex imaging revealed that EphA2 and CD137 are co-expressed in human tumors of high unmet medical need and therefore, using our highly modular Bicycle platform, we discovered BT7455, a Bicycle TICA that engages EphA2 and CD137 with high affinity, resulting in potent EphA2-dependent activity in vitro and robust anti-tumor activity in vivo with intermittent dosing in mouse models. Gene expression profiling of tumors revealed that BT7455 led to increased production of cytokines and chemokines known to drive T cell infiltration, the extent of which differentiated it from both a checkpoint inhibitor and an anti-CD137 monoclonal antibody agonist. BT7455 was well tolerated in preclinical species and liver function tests indicated no evidence of hepatic toxicity. Statement of Conclusions: In summary, we have identified EphA2 as a promising target to pair with a CD137 agonist. In addition to CD8+ T cells, tumor-resident dendritic cells are a likely contributor to anti-tumor immunity following treatment with Bicycle® TICAs, supporting utility for Bicycle® tumor-targeted CD137 agonists in solid tumors beyond those that are highly T cell-infiltrated. We have advanced a clinical development candidate, BT7455, to realize this potential for patients with EphA2-expressing cancers. Citation Format: Johanna Lahdenranta, Kristen Hurov, Heather Cohen, Lia Luus, Cara Bray, Peter Brown, Anna Devlen, Carly Campbell, Matthew Gray, Mike Kelly, Gemma Mudd, Punit Upadhyaya, Sailaja Battula, Kelvin Zhang, Anne-Sophie Dugast, Kevin McDonnell, Phil Brandish, Nicholas Keen. Tumor-targeted activation of CD137 using Bicycles: New insights into mechanism of action and discovery of BT7455, a clinical candidate for the treatment of EphA2-expressing cancers [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 5301.

Abstract 4642: Fatty acid synthase as a novel prognostic biomarker and a potential therapeutic target in diffuse malignant peritoneal mesothelioma

Abstract Diffuse malignant peritoneal mesothelioma (DMPM) is a rare and locally aggressive tumor with a dismal prognosis. The availability of new therapies and biomarkers for patients’ selection is still an urgent need. Altered lipid metabolism, which has been reported in several tumors, has not yet been studied in DMPM. By comparatively analyzing gene expression profiles of tumors form relapsed (n=18) and non-relapsed (n=18) DMPM patients, we initially found that fatty acid synthase (FASN) was up-modulated in relapsed tumors, with a significant linear correlation between FASN expression and shorter patient relapse-free survival (RFS), suggesting a potential role of FASN in DMPM progression. The prognostic significance of FASN was further assessed in a cohort of 80 DMPM patients who underwent cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy. Our results indicated that high FASN staining was significantly associated with reduced RFS (hazard ration (HR): 1.930; p=0.014) and Overall Survival (OS) (HR: 1.854; p=0.032) as assessed by immunohistochemistry in tissues microarray samples. The prognostic value of FASN, along with Ki67 staining and patient age, was also maintained in multivariate analysis in terms of both RFS (HR: 2.240; p=0.007) and OS (HR: 2.246; p=0.014). To validate FASN as a therapeutic target, we investigated the cytotoxic effects of non-selective (orlistat) and selective (cerulenin and C75) FASN inhibitors on patient-derived DMPM cell lines, both as single agents and in combination with selinexor -a selective inhibitor of the exportin chromosome maintenance protein. FASN pharmacological inhibition in DMPM cells resulted in a significant reduction of cell proliferation, especially upon C75 exposure. In combination experiments, we observed a synergistic activity of FASN inhibitors and selinexor, as determined by Chou and Talalay method. Through western blotting and flow cytometry analysis, we gained further insight into the mechanisms by which FASN inhibitors and selinexor synergize to reduce DMPM cell proliferation. Compared to single agents, the combined treatments resulted in increased apoptosis response and cell cycle impairment, as indicated by caspase-dependent apoptosis activation and cell accumulation in the G2-phase. Overall, our results support the relevance of FASN as a prognostic biomarker for DMPM patients and underscore its potential as a novel therapeutic target. Citation Format: Valentina Doldi, Silvia Martini, Chiara Maura Ciniselli, Alessia Beretta, Marcello Deraco, Shigeki Kusamura, Dario Baratti, Paolo Gandellini, Paolo Verderio, Nadia Zaffaroni. Fatty acid synthase as a novel prognostic biomarker and a potential therapeutic target in diffuse malignant peritoneal mesothelioma [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 4642.

Abstract 2855: ETS1, a target gene of the EWSR1::FLI1 fusion oncoprotein, regulates the expression of the focal adhesion protein TENSIN3

Abstract Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor that affects children and young adults. Ewing sarcomas harbor few mutations beyond the chromosomal translocation that initiates disease, and the mechanistic basis for the metastasis of these tumors remains poorly understood. The epigenome of EWS cells reflects the regulatory state of genes associated with the DNA binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG’s repression of transcription factor genes, particularly those encoding transcriptional regulators of cell differentiation. To examine EWSR1::FLI1/ERG’s regulation of gene expression, we assayed DNA binding using ChIP-seq or CUT&RUN, RNA using RNA-sequencing (RNA-seq) or qRT-PCR, and protein using immunoblotting or immunofluorescence. We depleted the expression of EWSR1::FLI1/ERG proteins using RNAi and overexpressed ETS1 using a full-length cDNA or CRISPR activation.RNA-seq analysis of control and EWSR1::FLI1-silenced TC-32, TC-71, and A673 EWS cells identified 67 genes encoding proteins with DNA binding activity that exhibited significant increases in expression following depletion of EWSR1::FLI1. Comparison of the expression of these 67 transcription factor genes in EWS cell lines (n=41) and tumors (n=79) showed 37 expressed at a wider range in tumors relative to cell lines (p <0.01). This more variable expression in EWS tumors suggests that one or more of these transcription factors could exert a phenotypic effect. Focusing on one of the EWSR1::FLI1-repressed target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3 repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1’s binding of promoter regions. Analysis of EWS cells in which we profiled ETS1 binding and assessed the effects of ectopically expressed ETS1 defined 265 genes as positively regulated by ETS1 of which 103 also exhibited an increase in expression following depletion of EWSR1::FLI1. Of these 103 genes, we focused on ETS1’s regulation of TNS3, as assessment of multiple EWS tumor expression profiles, showed positive correlation of ETS1 and TNS3 expression. TNS3 encodes TENSIN3, a focal adhesion protein that regulates cytoskeletal reorganization and contributes to cell migration by connecting the cytoplasmic tail of integrins to the actin cytoskeleton. EWS cell lines (SK-N-MC (EWSR1::FLI1) and ES-5838 (EWSR1::ERG), in which we activated ETS1 expression (CRISPRa) exhibited a migratory phenotype and increased TNS3 expression. Critically, the activated ETS1 EWS cell lines show TNS3 accumulation at leading cell edges, with F-actin cytoskeletal reorganization, a phenotype associated with cell migration. Our study demonstrates that EWS cells expressing ETS1 exhibit a more migratory phenotype, which, has the potential to promote the dissemination of cells and, thus, metastasis. Citation Format: Vernon Justice Ebegboni, Tamara L. Jones, Tayvia Brownmiller, Erica Pehrsson, Patrick Zhao, Soumya Sundara Rajan, Natasha J. Caplen. ETS1, a target gene of the EWSR1::FLI1 fusion oncoprotein, regulates the expression of the focal adhesion protein TENSIN3 [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 2855.

Stromal Signals Dominate Gene Expression Signature Scores That Aim to Describe Cancer Cell-intrinsic Stemness or Mesenchymality Characteristics.

Cancer self-renewal and migratory abilities are often characterized via gene module expression profiles, also called EMT or stemness gene expression signatures. Using published clinical tumor samples, cancer cell lines, and single cancer cells, we highlight the dominating influence of noncancer cells in low cancer cell content biopsies on their scores. We caution on their application for low cancer cell content clinical cancer samples with the intent to assign such characteristics or subtypes.

An extensive analysis of the prognostic and immune role of FOXO1 in various types of cancer.

Forkhead Box O1 (FOXO1) has been reported to play important roles in many tumors. However, FOXO1 has not been studied in pan-cancer. The purpose of this study was to reveal the roles of FOXO1 in pan-cancer (33 cancers in this study). Through multiple public platforms, a pan-cancer analysis of FOXO1 was conducted to obtained FOXO1 expression profiles in various tumors to explore the relationship between FOXO1 expression and prognosis of these tumors and to disclose the potential mechanism of FOXO1 in these tumors. FOXO1 was associated with the prognosis of multiple tumors, especially LGG (low grade glioma), OV (ovarian carcinoma), and KIRC (kidney renal clear cell carcinoma). FOXO1 might play the role of an oncogenic gene in LGG and OV, while playing the role of a cancer suppressor gene in KIRC. FOXO1 expression had a significant correlation with the infiltration of some immune cells in LGG, OV, and KIRC. By combining FOXO1 expression and immune cell infiltration, we found that FOXO1 might influence the overall survival of LGG through the infiltration of myeloid dendritic cells or CD4+ T cells. Functional enrichment analysis and gene set enrichment analysis showed that FOXO1 might play roles in tumors through immunoregulatory interactions between a lymphoid and a non-lymphoid cell, TGF-beta signaling pathway, and transcriptional misregulation in cancer. FOXO1 was associated with the prognosis of multiple tumors, especially LGG, OV, and KIRC. In these tumors, FOXO1 might play its role via the regulation of the immune microenvironment.

Predicting drug response from single-cell expression profiles of tumours

BackgroundIntra-tumour heterogeneity (ITH) presents a significant obstacle in formulating effective treatment strategies in clinical practice. Single-cell RNA sequencing (scRNA-seq) has evolved as a powerful instrument for probing ITH at the transcriptional level, offering an unparalleled opportunity for therapeutic intervention.ResultsDrug response prediction at the single-cell level is an emerging field of research that aims to improve the efficacy and precision of cancer treatments. Here, we introduce DREEP (Drug Response Estimation from single-cell Expression Profiles), a computational method that leverages publicly available pharmacogenomic screens from GDSC2, CTRP2, and PRISM and functional enrichment analysis to predict single-cell drug sensitivity from transcriptomic data. We validated DREEP extensively in vitro using several independent single-cell datasets with over 200 cancer cell lines and showed its accuracy and robustness. Additionally, we also applied DREEP to molecularly barcoded breast cancer cells and identified drugs that can selectively target specific cell populations.ConclusionsDREEP provides an in silico framework to prioritize drugs from single-cell transcriptional profiles of tumours and thus helps in designing personalized treatment strategies and accelerating drug repurposing studies. DREEP is available at https://github.com/gambalab/DREEP.

Retracted: Screening Tumor-Related Genes of Gallbladder Cancer Based on AR-Based Tumor Expression Profile Gene Chip

Retracted: Screening Tumor-Related Genes of Gallbladder Cancer Based on AR-Based Tumor Expression Profile Gene Chip

Epigenetic Profiles of Triple-Negative Breast Cancers of African American and White Females

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and appears to have disproportionately higher incidence and worse outcomes among younger African American females. To investigate whether epigenetic differences exist in TNBCs of younger African American females that may explain clinical disparities seen in this patient group. This cross-sectional study used clinical, demographic, DNA methylation (HumanMethylation450; Illumina), and gene expression (RNA sequencing) data for US patient populations from publicly available data repositories (The Cancer Genome Atlas [TCGA], 2006-2012, and Gene Expression Omnibus [GEO], 2004-2013) accessed on April 13, 2021. White and African American females with TNBC identified in TCGA (69 patients) and a validation cohort of 210 African American patients from GEO (GSE142102) were included. Patients without available race or age data were excluded. Data were analyzed from September 2022 through April 2023. DNA methylation and gene expression profiles of TNBC tumors by race (self-reported) and age were assessed. Age was considered a dichotomous variable using age 50 years as the cutoff (younger [<50 years] vs older [≥50 years]). A total of 69 female patients (34 African American [49.3%] and 35 White [50.7%]; mean [SD; range] age, 55.7 [11.6; 29-82] years) with TNBC were included in the DNA methylation analysis; these patients and 210 patients in the validation cohort were included in the gene expression analysis (279 patients). There were 1115 differentially methylated sites among younger African American females. The DNA methylation landscape on TNBC tumors in this population had increased odds of enrichment of hormone (odds ratio [OR], 1.82; 95% CI, 1.21 to 2.67; P = .003), muscle (OR, 1.85; 95% CI, 1.44 to 2.36; P < .001), and proliferation (OR, 3.14; 95% CI, 2.71 to 3.64; P < .001) pathways vs other groups (older African American females and all White females). Alterations in regulators of these molecular features in TNBCs of younger African American females were identified involving hormone modulation (downregulation of androgen receptor: fold change [FC] = -2.93; 95% CI, -4.76 to -2.11; P < .001) and upregulation of estrogen-related receptor α (FC = 0.86; 95% CI, 0.34 to 1.38; P = .002), muscle metabolism (upregulation of FOXC1: FC = 1.33; 95% CI, 0.62 to 2.03; P < .001), and proliferation mediators (upregulation of NOTCH1: FC = 0.71; 95% CI, 0.23 to 1.19; P = .004 and MYC (FC = 0.81; 95% CI, 0.18 to 1.45; P = .01). These findings suggest that TNBC of younger African American females may represent a distinct epigenetic entity and offer novel insight into molecular alterations associated with TNBCs of this population. Understanding these epigenetic differences may lead to the development of more effective therapies for younger African American females, who have the highest incidence and worst outcomes from TNBC of any patient group.

P10.23.A A PATIENT-DERIVED FUNCTIONAL GBM DRUG SCREEN OFFERS A TOOL TO PREDICT TMZ RESPONSE AND ASSESS SENSITIVITY TO CANDIDATE TREATMENTS ON A PER PATIENT BASIS

Abstract BACKGROUND Major obstacles that have impeded the development of effective new therapies for GBM include inter- and intratumoral heterogeneity, the blood-brain-barrier and use of sub-optimal cell line-based preclinical models. Taking these hurdles into account, we have set up a patient-derived GBM drug-screening platform. Molecular similarities and dissimilarities between tumors and derived cultures were investigated and the predictive power of our assay for patient response to TMZ was assessed. A large panel of GBM cells was screened for sensitivity to available oncological agents with the aim of deriving a set of drugs with favorable physicochemical properties for BBB crossing and potent activity in (a subset of) GBM cultures. Finally, we determined the success rate of performing a small-scale screen with 20 selected agents within 4 weeks post-surgery. MATERIAL AND METHODS Tissue samples as well as ultrasonic surgical aspirates (USA) were received from GBM surgeries and utilized to establish glioma stem cell (GSC) cultures. Correspondence of GSCs to parental tumors was assessed by DNA/RNA-sequencing. In vitro sensitivity to temozolomide was screened across 55 GSC cultures. Cell viability readouts were related to clinical parameters of corresponding patients. FDA-approved anticancer agents (n=107) were screened on 45 GBM cultures by dose-response testing and viability was assessed using Cell Titer-Glo. Twenty selected agents were screened on 24 fresh tumor samples within 4 weeks of receiving the tissue. RESULTS By combining both USA and tissue piece-derived dissociation protocols, culture success increased to 95%, ensuring representation of the near-complete spectrum of GBM subtypes. Tumor DNA and RNA sequences revealed strong similarities to corresponding GSC cultures although differences related to neuronal signaling, immune interactions, and cell cycle were observed. Tumor expression profiles of hallmark genes of GBM were faithfully retained in derived cell cultures. In vitro screening of TMZ on a large cohort identified 3 response categories (responders/intermediates/non-responders) for which Cox regression analysis revealed significantly different overall survival curves of corresponding patients. Screening of approved anticancer agents on 45 GBM cultures underscored the tremendous intertumoral heterogeneity in drug sensitivities. We identified 20 potent agents each effective at clinically-achievable concentrations in (a subset of) GBM cultures with favorable BBB penetration properties (CNS-MPO score). Screening of these agents on a per patient basis within 4 weeks of receiving tissue was successful in 18 out of 24 (75%) tested tumors. CONCLUSION Patient-derived functional drug screening offers a tool to predict TMZ response in GBM patients and assess tumor sensitivity to candidate treatments, either for GBM subsets or on a per patient basis.

Associations between prediagnostic aspirin use and ovarian tumor gene expression.

Aspirin use has been associated with reduced ovarian cancer risk, yet the underlying biological mechanisms are not fully understood. To gain mechanistic insights, we assessed the association between prediagnosis low and regular-dose aspirin use and gene expression profiles in ovarian tumors. RNA sequencing was performed on high-grade serous, poorly differentiated, and high-grade endometrioid ovarian cancer tumors from the Nurses' Health Study (NHS), NHSII, and New England Case-Control Study (n = 92 cases for low, 153 cases for regular-dose aspirin). Linear regression identified differentially expressed genes associated with aspirin use, adjusted for birth decade and cohort. False discovery rates (FDR) were used to account for multiple testing and gene set enrichment analysis was used to identify biological pathways. No individual genes were significantly differentially expressed in ovarian tumors in low or regular-dose aspirin users accounting for multiple comparisons. However, current versus never use of low-dose aspirin was associated with upregulation of immune pathways (e.g., allograft rejection, FDR = 5.8 × 10-10 ; interferon-gamma response, FDR = 2.0 × 10-4 ) and downregulation of estrogen response pathways (e.g., estrogen response late, FDR = 4.9 × 10-8 ). Ovarian tumors from current regular aspirin users versus never users were also associated with upregulation in interferon pathways (FDR <1.5 × 10-4 ) and downregulation of multiple extracellular matrix (ECM) architecture pathways (e.g., ECM organization, 4.7 × 10-8 ). Our results suggest low and regular-dose aspirin may impair ovarian tumorigenesis in part via enhancing adaptive immune response and decreasing metastatic potential supporting the likely differential effects on ovarian carcinogenesis and progression by dose of aspirin.

Abstract 2437: HMOX1 modulates tumor stemness and metastatic properties in prostate cancer

Abstract Eighty percent of prostate cancers (PCa) metastasize to bone, showing a significant source of patient morbidity. PCa stem-like cells (PCSCs) are capable to quiescently outlast retaining the ability to proliferate and regenerate, consequently remaining able to develop therapy-resistant tumors and metastatic lesions. We have previously reported that the overexpression of heme oxygenase 1 (HO-1), the rate limiting enzyme in heme degradation, leads to a less aggressive PCa phenotype. However, its effect on metastasis-stemness (MS) remains unknown. In this work, we address the biological significance of HO-1 in association with relevant MS genes for PCa progression. Clonogenic assays performed in PCa cells (PC3 and C4-2B) to assess colony formation, evidenced a reduction on the stem-like properties of tumor cells treated with hemin (FDA approved drug and specific HO-1 inducer). RNA-seq analysis to identify MS genes that could be modulated by HO-1 induction, revealed 32 MS-genes that were modulated in PC3-hemin vs. PC3-control cells. We then mined the Oncomine database (n = 1,128) and found that 15/32 HO-1-modulated MS genes were significantly dysregulated in PCa compared with normal gland. To extend our findings, further computational analyses were conducted, and a custom-made bioinformatics tool (Gene Hunter) was created to analyze gene expression and clinicopathological profiles across multiple publicly available PCa datasets with normal, adjacent, tumoral and metastatic samples (n = 1,629). We found that ADAM15, BCL2L1, LTBR, MBNL2 and SPINT1 are consistently dysregulated across different comparisons, with expression profiles in tumors that could be reverted by hemin treatment, according to our RNA-seq results. We constructed a risk score that efficiently stratified patients in high, intermediate and low-risk of biochemical-relapse groups (p&amp;lt;0.01). We also performed multiple survival analyses including, overall, progression-free, relapse-free and metastasis-free survival data. We found that high MBNL2, a gene previously reported to be related to embryonic stem cell differentiation and to antimetastatic effects in other tumors, was associated with lower risk of relapse, progression and metastasis (p&amp;lt;0.05). Of note, the expression of MBNL2 was downregulated in PCa samples compared with normal gland or normal adjacent tissue. Interestingly MBNL2 expression can be reverted by HO-1 induction in PCa cells, as validated by RT-qPCR. Accordingly, those patients with high expression of MBNL2 showed a decreased risk of biochemical-relapse when they presented high HMOX1 levels (HR = 0.4615, p = 0.0186). Altogether, we highlight the relevance of HO-1 in modulating MS-associated genes in PCa and point out to MBNL2 as a potential druggable target for disease intervention. Citation Format: Agustina A. Sabater, Ayelen R. Toro, Pablo A. Sanchis, Gaston M. Pascual, Rocio A. Seniuk, Elba S. Vazquez, Javier Cotignola, Juan A. Bizzotto, Sofia Lage-Vickers, Geraldine Gueron. HMOX1 modulates tumor stemness and metastatic properties in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2437.

Abstract 3523: A systematic comparison of molecular features shared by H3K27-altered diffuse midline gliomas and posterior fossa A ependymomas

Abstract In children, brain tumors account for the highest mortality rates of all malignancies, and in contrast to adult brain cancers, pediatric tumors often arise in infratentorial regions. Two tumors in particular, H3K27-altered diffuse midline gliomas (DMGs) and group-A posterior fossa ependymomas (PFAs) arise from structures around the fourth ventricle, and strikingly, both tumors exhibit a global decrease in the histone mark histone 3, lysine 27 tri-methylation (H3K27me3) that is associated with transcriptional repression. H3K27-altered DMGs primarily are found primarily in the pons and exhibit by lysine-to-methionine mutations at H3K27 (H3K27M). This missense mutation results in inhibition of the catalytic activity of the polycomb repressive complex 2 (PRC2) which mediates H3K27me3 deposition. PFAs are found in the lateral recess of the fourth ventricle and the cerebellum and demonstrate overexpression of EZH inhibitory protein (EZHIP). EZHIP contains a domain with striking resemblance to the mutant H3K27M histone tail and similarly blocks PRC2 activity. Although H3K27me3 levels are dramatically reduced in both areas, select genes retain H3K27me3 in both tumor types. Increasingly, data have emerged demonstrating that cases of PFAs actually harbor H3K27M mutations and cases of H3-wildtype DMGs in fact overexpress EZHIP. The neighboring origins and overlapping recurrent H3K27me3-modulating alterations observed in these two pediatric brain tumors suggest certain populations of cells in the developing hindbrain may be particularly prone to malignant transformation in the setting of PRC2 inhibition. A systematic comparison of genomic, transcriptomic, and epigenomic datasets for both tumors identified key molecular similarities between subsets of DMGs and PFAs and highlighted how developmental signatures may be preserved in tumor expression profiles. Specifically, these analyses identify shared copy-number profiles between PFAs and different subtypes of DMGs and demonstrate that the Activin A Receptor Type 1 (ACVR1), a gene with recurrent activating mutations in H3K27-altered DMGs, is overexpressed in a subset PFAs with worse survival outcomes. Additionally, interrogation of the H3K27me3 and enhancer landscapes identified critical genes with similar chromatin profiles and revealed heterogeneity in repression of H3K27me3-marked genes that correlated with patterns of inferred anatomic origins for each tumor. Moreover, one of these genes, cellular retinoic acid binding partner 1 (CRABP1) showed enrichment in progenitor cell types in single-cell transcriptomic datasets. Together, these analyses characterize the common and unique molecular features of H3K27-altered DMGs and PFAs and shed light on the cellular contexts of the developing hindbrain that lead to development of each tumor. Citation Format: Matthew Pun, Drew Pratt, Patricia R. Nano, Piyush K. Joshi, Li Jiang, Bernhard Englinger, Arvind Rao, Marcin Cieslik, Arul M. Chinnaiyan, Kenneth Aldape, Stefan Pfister, Mariella G. Filbin, Aparna Bhaduri, Sriram Venneti. A systematic comparison of molecular features shared by H3K27-altered diffuse midline gliomas and posterior fossa A ependymomas. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3523.

Structural and functional properties of mSWI/SNF chromatin remodeling complexes revealed through single-cell perturbation screens

The mammalian SWI/SNF (mSWI/SNF or BAF) family of chromatin remodeling complexes play critical roles in regulating DNA accessibility and gene expression. The three final-form subcomplexes-cBAF, PBAF, and ncBAF-are distinct in biochemical componentry, chromatin targeting, and roles in disease; however, the contributions of their constituent subunits to gene expression remain incompletely defined. Here, we performed Perturb-seq-based CRISPR-Cas9 knockout screens targeting mSWI/SNF subunits individually and in select combinations, followed by single-cell RNA-seq and SHARE-seq. We uncovered complex-, module-, and subunit-specific contributions to distinct regulatory networks and defined paralog subunit relationships and shifted subcomplex functions upon perturbations. Synergistic, intra-complex genetic interactions between subunits reveal functional redundancy and modularity. Importantly, single-cell subunit perturbation signatures mapped across bulk primary human tumor expression profiles both mirror and predict cBAF loss-of-function status in cancer. Our findings highlight the utility of Perturb-seq to dissect disease-relevant gene regulatory impacts of heterogeneous, multi-component master regulatory complexes.