Abstract A29: Multiplex functional genomics assay to deconvolute compound genetic drivers of bladder cancer

Abstract

Abstract Next-generation sequencing has led to substantial insights into the genetic and molecular heterogeneity of bladder cancer. However, the functional consequences of many of these genetic abnormalities have not been explored. Current genetically defined models of tumorigenesis are poorly suited for systematic functional interrogation of the diverse genetic abnormalities in bladder cancer. To address this issue, we are developing an approach that is readily scalable and leverages an established organoid transformation assay, multiplex transduction of organoids by a lentiviral pool, and digital profiling by single-cell sequencing. We found that C57BL/6J mouse basal urothelial cells (Lin−EpCAM+CD49fhigh) efficiently initiated organoids in 3D culture in defined media conditions. Urothelial organoids were readily transduced with lentivirus to achieve viral copy numbers (VCNs) of up to 20 with a linear relationship between VCN and lentiviral dose. As lentiviral transduction follows a Poisson distribution, we reasoned that a lentiviral pool could be used to introduce random combinations of genetic alterations into urothelial organoids. We selected 33 recurrently altered genes in bladder cancer and categorized them as gain of function (genomic amplification and activating mutations) and loss of function (genomic deletion and inactivating mutations). We cloned lentiviral constructs that express open reading frames to recapitulate gain-of-function events and short hairpin RNAs to model loss-of-function events. Each lentiviral clone has also been barcoded with matching 10-nucleotide sequences at both the 5' and 3' ends to facilitate barcode enumeration by DNA amplicon sequencing and RNA sequencing. In preliminary experiments, we have transduced mouse urothelial organoids with the barcoded lentiviral pool to target a mean viral copy number of 4. After a brief period of culture, organoids were grafted subcutaneously into immune-deficient mice to biologically select for tumorigenic clones. Resultant tumors will be isolated for single-cell DNA amplicon sequencing to enable the enumeration of combinations of oncogenic events within each tumor clone that induce bladder cancer tumorigenesis. Optimization of single-cell RNA sequencing is ongoing to allow direct, massively parallel association of genotype to bladder cancer phenotype within this assay. Overall, we are establishing a functional cancer genomics platform to provide rapid functional annotation of genetic alterations and their roles in bladder cancer initiation. Another goal of these studies is the generation of multiple genetically defined models of bladder cancer that recapitulate the spectrum of human disease. Citation Format: Alicia Wong, Huiyun Sun, Sujata Jana, Andrew C. Hsieh, John K. Lee. Multiplex functional genomics assay to deconvolute compound genetic drivers of bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A29.