Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells

Abstract

We used synthetic lethal high-throughput screening to interrogate 23,550 compounds for their ability to kill engineered tumorigenic cells but not their isogenic normal cell counterparts. We identified known and novel compounds with genotype-selective activity, including doxorubicin, daunorubicin, mitoxantrone, camptothecin, sangivamycin, echinomycin, bouvardin, NSC146109, and a novel compound that we named erastin. These compounds have increased activity in the presence of hTERT, the SV40 large and small T oncoproteins, the human papillomavirus type 16 (HPV) E6 and E7 oncoproteins, and oncogenic HRAS. We found that overexpressing hTERT and either E7 or LT increased expression of topoisomerase 2alpha and that overexpressing RAS(V12) and ST both increased expression of topoisomerase 1 and sensitized cells to a nonapoptotic cell death process initiated by erastin.

Ferroptosis: process and function

“…First identified as a ferroptosis inducer in 2003, erastin was found to be synthetic lethal with expression of the engineered mutant Ras oncogene in human foreskin fibroblasts (BJeLR), but not their isogenic primary counterparts. 2 Ras-selective lethal small molecule (RSL)-3 and RSL5 were later identified in 2008 in another high-throughput small molecule-screening study that selectively killed BJeLR cells in a non-apoptotic manner. 3 Inhibition of apoptosis, necrosis, necroptosis, and autophagy by small molecule inhibitors (e.g., Z-VAD-FMK, BOC-D-FMK, wortmannin, and necrostatin-1) cannot reverse RSL-induced cell death (Table 2).…”

“…1,2,4 Ferroptotic cells exhibit changed mitochondrial morphology and cristae structure. Smaller than normal mitochondria with increased mitochondrial membrane density and reduction/vanishing of mitochondria crista have been observed in ferroptosis following erastin treatment in BJeLR cells.…”

“…Smaller than normal mitochondria with increased mitochondrial membrane density and reduction/vanishing of mitochondria crista have been observed in ferroptosis following erastin treatment in BJeLR cells. 1,2,4 Induction of ferroptosis by genetic inactivation of GPX4 in immortalized fibroblasts and kidney tissue has been associated with outer mitochondrial membrane rupture, as observed using transmission electron microscopy. 5 In contrast, the structural integrity of the nucleus is retained following erastin treatment in cancer cells.…”

“…5 In contrast, the structural integrity of the nucleus is retained following erastin treatment in cancer cells. 2 No nuclear condensation or chromatin margination are observed following erastin treatment in cancer cells. 2 These morphological features help us distinguish ferroptosis from apoptosis, necroptosis, and autophagy (Table 1).…”

Systematic interpretation of genetic interactions using protein networks

“…Genetic interactions have been used extensively to shed light on pathway organization in model organisms [1][2][3][4] . In humans, genetic interactions are critical in linkage analysis of complex diseases 5 and in discovery of new pharmaceuticals 6 . Although genetic interactions are classically identified by mutant screens 7 , recent studies have applied systematic 'reverse' methods such as synthetic genetic arrays (SGA) 8 or synthetic lethal analysis by microarrays (SLAM) 9 to catalog ~4,000 synthetic-lethal and synthetic-sick interactions in Saccharomyces cerevisiae.…”