A functional genomics process for systematic dissection and mutation-specific target discovery in breast cancer PIK3CA hotspot mutations [CRISPR KO Screen Data]
Despite the general understanding that different mutations impart different phenotypic changes within a cell, the majority of targeted therapies in cancer treatment are used to treat all mutations in the same gene regardless of location or phenotypic effects. There is a significant unmet need to distinguish distinct changes in cellular signaling which may provide opportunities for mutation-specific treatment with reduced toxicity to patients. Herein, we describe a series of functional genomic analysis with a unique isogenic cell line panel to accurately identify targetable differences between mutations within the same gene.  Using an isogenic cell line model bearing two distinct hotspot PIK3CA mutations found in breast cancer, we were able to identify many gene expression and chromatin accessibility differences. These findings allowed us to identify mutation specific molecular targets, specifically AREG as well as a proximal gene regulatory region, that may provide clinically relevant targets. When disrupted, these targets induce a mutation-specific decrease in proliferation and survival in vivo. These findings suggest new mutation-specific modes of treatment for PIK3CA mutant breast cancer and provide a means with which to find mutation-specific targets for the treatment of other oncogenic mutations. Overall design: A modified CRISPR screen was performed with a select cohort of gene targets selected based on two criteria: 1. genes exhibited significantly increased RNA expression (log2 fold change greater than 1.5 and p-adjusted value less than 0.05) in either PIK3CA-mutant cell lines and 2. gene demonstrated significantly increased accessibility in either mutant cell line. Differential accessibility was performed using DESeq2 and nearest neighbor annotation was performed using ChIPseeker (version 1.30.3) with the default annotation conditions of +/- 3kb from the TSS. Based on this selection criteria, 312 genes were selected, for which 280 guides were available in the Brunello whole genome single guide RNA (sgRNA) library. The Brunello whole genome sgRNA library was modified for these 280 genes and prepared by the Vanderbilt Functional Genomics core in the lentiCRISPRv2 plasmid background (Full list of guides Mageck_Guide.csv). MCF-10A cells were cultured in the maintenance media conditions at a density of 500,000 cells/well in a 6-well plate. 24 hours after seeding, cells were infected with viral supernatant in maintenance media containing 5 ug/mL polybrene. 24 hours post-infection cells were placed in selection media containing 1 ug/mL of puromycin and maintained for two weeks. Following two weeks of selection, libraries were prepared and sequenced following the protocol described in Sanjana et al. Libraries were sequenced by the VANTAGE core and analysis was performed using the maximum likelihood estimation (MLE) algorithm within the MAGeCK software package(version 0.5.9.5).