Human Haploid Cell Genetics Reveals Roles for Lipid Metabolism Genes in Nonapoptotic Cell Death

Abstract

Little is known about the regulation of nonapoptotic cell death. Using massive insertional mutagenesis of haploid KBM7 cells we identified nine genes involved in small-molecule-induced nonapoptotic cell death, including mediators of fatty acid metabolism (ACSL4) and lipid remodeling (LPCAT3) in ferroptosis. One novel compound, CIL56, triggered cell death dependent upon the rate-limiting de novo lipid synthetic enzyme ACC1. These results provide insight into the genetic regulation of cell death and highlight the central role of lipid metabolism in nonapoptotic cell death.

Figure 1

Effect of lethal molecules in KBM7 cells. (a) Structure of the lethal molecules tested in this study. (b) Dose–response analysis of seven lethal compounds in KBM7 cells ± three ferroptosis inhibitors ferrostatin-1 (Fer-1), beta-mercaptoethanol (β-ME), and ciclopirox olamine (CPX). Norm. AB Fluor.: Normalized Alamar Blue Fluorescence, an indication of cell viability. Data represents the mean ± SD from three independent biological replicates.

Figure 2

Identifying genetic determinants of small molecule lethality. (a–e) Circos Plots for each lethal compound showing every gene with at least one mapped retroviral insertion represented as a blue circle oriented toward the chromosomal position of the gene. Circle size is proportional to the number of insertions, and distance from the center is proportional to the significance (p-value) of this number of insertions relative to control cells. Genes enriched in selected versus unselected pools with p-values < 10^–3 are indicated.

Figure 3

Analysis of the role of ACACA in CIL56-induced death. (a) The role of ACC1 (encoded by ACACA) in fatty acid synthesis. FAS: fatty acid synthase. (b) Western blot of ACC1 levels in two independent HT-1080 cell clones (H and P). Each lysate was loaded twice in adjacent lanes. (c–f) Viability of HT-1080 cells and variants lacking ACC1 treated with various lethal compounds. (g) Viability of HT-1080 cells treated for 24 h with lethal compounds ± TOFA (5 μM) or Fer-1 (1 μM). Norm. AB Fluor.: Normalized Alamar Blue Fluorescence, an indication of cell viability. (h–j) Metabolomic analysis of HT-1080 cells treated with CIL56 (6.5 μM) ± TOFA (4 μM). (h) Fold-change in abundance of 144 metabolites significantly altered by CIL56 treatment (FDR q < 0.01) ± TOFA. (i) Fold-change in metabolite levels for saturated fatty acids. (j) Fold-change in metabolite levels for polyunsaturated fatty acids.