A Gene Expression High-Throughput Screen (GE-HTS) for Coordinated Detection of Functionally Similar Effectors in Cancer

Abstract

Genome-wide, loss-of-function screening can be used to identify novel vulnerabilities upon which specific tumor cells depend for survival. Functional Signature Ontology (FUSION) is a gene expression-based high-throughput screening (GE-HTS) method that allows researchers to identify functionally similar proteins, small molecules, and microRNA mimics, revealing novel therapeutic targets. FUSION uses cell-based high-throughput screening and computational analysis to match gene expression signatures produced by natural products to those produced by small interfering RNA (siRNA) and synthetic microRNA libraries to identify putative protein targets and mechanisms of action (MoA) for several previously undescribed natural products. We have used FUSION to screen for functional analogues to Kinase suppressor of Ras 1 (KSR1), a scaffold protein downstream of Ras in the Raf-MEK-ERK kinase cascade, and biologically validated several proteins with functional similarity to KSR1. FUSION incorporates bioinformatics analysis that may offer higher resolution of the endpoint readout than other screens which utilize Boolean outputs regarding a single pathway activation (i.e., synthetic lethal and cell proliferation). Challenges associated with FUSION and other high-content genome-wide screens include variation, batch effects, and controlling for potential off-target effects. In this review, we discuss the efficacy of FUSION to identify novel inhibitors and oncogene-induced changes that may be cancer cell-specific as well as several potential pitfalls within FUSION and best practices to avoid them.

KSR1- and ERK-dependent translational regulation of the epithelial-to-mesenchymal transition

“…The analysis was performed using 2 -ΔΔC T method ( Schmittgen and Livak, 2008 ). For polysome gradients, the RNA levels were quantified from the cDNA using the standard curve method, summed across all fractions ( Kortum and Lewis, 2004 ; Nguyen et al, 2002 ; Fisher et al, 2011 ; Fisher et al, 2015 ; Morrison et al, 2009 ; Rao et al, 2020 ) and presented as a percentage of the total fractions.…”

“…The analysis was performed using 2 -ΔΔC T method (79). For polysome gradients, the RNA levels were quantified from the cDNA using the standard curve method, summed across all fractions (3)(4)(5)(6)(7)(8) and presented as a percentage of the total fractions.…”

“…Kinase Suppressor of Ras 1 (KSR1) serves as a scaffold for Raf, MEK, and ERK enabling the efficient transmission of signals within the mitogen activated protein kinase (MAPK) cascade ( Kortum and Lewis, 2004 ; Nguyen et al, 2002 ). Although KSR1 is dispensable for normal development, it is necessary for oncogenic Ras-induced tumorigenesis including colorectal cancer cells ( Kortum and Lewis, 2004 ; Nguyen et al, 2002 ; Fisher et al, 2011 ; Fisher et al, 2015 ; Morrison et al, 2009 ; Rao et al, 2020 ), suggesting that KSR1 may modulate aberrant signals that redirect the function of effectors typically involved in normal cellular homeostasis. Activating Ras mutations are present in over 40 % of colorectal cancers (CRC), and associated with advanced disease and decreased overall survival ( Haigis, 2017 ; Serebriiskii et al, 2019 ).…”

Pan-Cancer Analysis of Human Kinome Gene Expression and Promoter DNA Methylation Identifies Dark Kinase Biomarkers in Multiple Cancers

“…Recent structural studies have also focused on Tyr pseudokinases such as ROR1 to understand their mechanism and target them pharmacologically [ 15 ]. Other high-throughput screening methods to identify functional analogs to pseudokinase, Kinase suppressor of Ras 1 (KSR1) (involved in EGFR-Ras-MAP kinase pathway) have been recently published [ 16 ].…”

The Study of Cancer Susceptibility Genes

“…Consistent with the previous study, upregulated genes belong to the DNA repair group and include genes for DNA helicases, chromosome maintenance complexes, recombination, and replication. The Functional Signature Ontology (FUSION) approach of genome-wide, loss-of-function screening is another way to identify critical genes in cancer and find vulnerabilities of tumor cells [ 4 ]. A gene expression-based high-throughput screening method allows researchers to identify novel therapeutic targets.…”