Organoid Imaging Strategies for Accelerating Translational Research

<h3>Background</h3> Uveal melanoma (UM) is a rare tumor characterized by mutually exclusive activating mutations in GNAQ in GNA11, followed by secondary events in BAP1, SF3B1 and EIF1AX. Notably, a large subset of patients presents with copy-loss of chromosome 3 (monosomy 3), which is highly associated with metastatic progression in late-stage disease. Monosomy 3 tumors demonstrate a marked resistance to chemotherapy, targeted therapeutics, and immunotherapy, despite successes observed in cutaneous melanoma. Informing on the biology underlying chromosome 3 copy-loss, and its impact on the tumor microenvironment, is critical towards directing future efforts in targeted therapeutics and immunotherapy. Current limited insight can be attributed to both lack of: (a) preclinical models, and (b) in-depth characterization of paired primary and metastatic tumors in patients. <h3>Methods</h3> To address this, we first induced chromosome 3 copy-loss through CRISPR-based centromere targeting in a well characterized Disomy 3 UM cell line. Disomy 3 (D3) and Monosomy 3 (M3) clones derived from these efforts have enabled us to develop patient derived xenograft (PDX) models to compare D3 and M3 behavior in paired primary and metastatic settings. Additionally, we identified and collected a range of match-paired (primary and metastatic) clinical UM samples across multiple patients. Leveraging these unique samples, we applied spatial transcriptomics to inform on tumor intrinsic and extrinsic features of progressive UM, identifying gene signatures of disease advancement and deconvoluting the evolving tumor microenvironment. <h3>Results</h3> Investigation of metastatic UM tumor heterogeneity in our models enables us to characterize unique features of phenotypically transformed clones (e.g. depigmentation and growth advantage) (figure 1). We also adapted existing methodology to infer chromosomal copy number events from spatial transcriptomics data to our PDX system, overcoming the lack of same-species microenvironment controls. We complement our preclinical analyses with an investigation of spatial heterogeneity in a patient cohort of paired primary and metastatic tumors (figure 2). Leveraging single-cell deconvolution in this paired dataset, we captured unique immune microenvironments in primary vs. metastatic tumors. Additionally, we integrated our preclinical tumor intrinsic signatures to pair differential gene expression signatures of tumor sub-clones with differential immune cell populations. <h3>Conclusions</h3> Our methodology allows for deep characterization of sub-clonal heterogeneity in primary and metastatic settings and informs on the unique microenvironmental heterogeneity underlying invasiveness and outgrowth of M3 tumors. More broadly, comparing these preclinical and patient tumors provides an opportunity to expand on our knowledge of metastatic disease drivers and derive prognostic signatures associated with poor survival and lack of response to immunotherapy. 

1498 Deploying spatial transcriptomics to inform on intratumoral heterogeneity in late-stage uveal melanoma leveraging advanced preclinical modeling and clinical samples


 Uveal Melanoma (UM) is a rare tumor characterized by mutations in GNAQ or GNA11, followed by a mutation in BAP1, SF3B1 or EIF1AX. Notably, a large subset of BAP1 mutant patients present with a unique chromosome 3 copy-number (CN) loss and higher likelihood of metastases. The biology underlying the causes and consequences of Monosomy 3 (M3), and its role in metastases is largely unexplored. This can be partially attributed to the relatively few numbers of preclinical models of UM, and the inability to delineate M3 from BAP1 loss. To address this, we derived a preclinical isogenic model of chromosome 3 CN loss through CRISPR-based centromere targeting in a well characterized UM cell line lacking a BAP1 mutation. Clones with and without chromosome 3 loss were derived from these efforts. Recent studies have evaluated genomic events in primary UM and described five CN aberration-based subtypes expanding beyond M3, with the fifth subtype being called the “ultra-high-risk UM” based on poor survival and extremely high metastatic rate. Whole genome sequencing across our clones confirmed chromosome 3 CN loss, and in addition to this, recapitulated all of the core genomic features characteristic of the ultra-high-risk UM group. In comparison, our models without chromosome 3 CN loss maintained a genomic profile more characteristic of lower risk UM subtypes. The genomic landscape of our isogenic low risk (LR) and high risk (HR) clones suggest that chromosome 3 CN loss alone in the absence of BAP1 mutations can be the driver for acquisition of downstream genomic events seen in aggressive UM. Further, using single cell DNA sequencing on the parental model, LR and HR clones, we confirmed that chromosome 3 CN loss in our clones was engineered, and not a pre-existing subclone in the parental cell line. Bulk RNAseq on our LR and HR clones demonstrate concordance with genomic regions of losses and gains, while also highlighting upregulation of pathways such as cell cycle and DNA damage response in the HR clones. Next, we developed patient derived xenograft (PDX) models demonstrating differential behavior in paired in vivo settings, where HR clones had higher fitness advantage and metastatic potential, specifically to the mouse liver. This modeling has enabled us to employ spatial transcriptomics in a PDX setting, adapt existing methodology to infer chromosomal copy number events in our system and inform transcriptional and genomic phenotypes akin to the aggressive HR clones. Thus, using our engineered preclinical models, we demonstrate that chromosome 3 loss, in the absence of BAP1 mutations is capable of driving aggressive disease in UM, characterized by genomic evolution, transcriptional reprogramming and in vivo invasiveness.
 Citation Format: Sanjana Srinivasan, Johnathon Rose, Rosalba Minelli, Christopher Terranova, Parth Shah, Joseph R. Daniele, Michael Peoples, Meng He, Chiu-Yi Liu, Jason Gay, Melinda Soeung, Sanjay Soni, Fangyang Wang, Christopher Bristow, Khalida Wani, Luigi Perelli, Dmitry Loza, Ningping Feng, Maxime Tarabichi, Peter Van Loo, Andrew Futreal, Scott Woodman, Alexander Lazar, Christopher P. Vellano, Joseph Marszalek, Giulio F. Draetta, Timothy Heffernan, Alessandro Carugo, Giannicola Genovese, Virginia Giuliani. Chromosome 3 copy number loss alone drives tumor evolution to aggressive disease in 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 1758.

Abstract 1758: Chromosome 3 copy number loss alone drives tumor evolution to aggressive disease in uveal melanoma

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Organoid Imaging Strategies for Accelerating Translational Research

1498 Deploying spatial transcriptomics to inform on intratumoral heterogeneity in late-stage uveal melanoma leveraging advanced preclinical modeling and clinical samples

Abstract 1758: Chromosome 3 copy number loss alone drives tumor evolution to aggressive disease in uveal melanoma