Copper induces cell death by targeting lipoylated TCA cycle proteins

Abstract

Copper is an essential cofactor for all organisms, and yet it becomes toxic if concentrations exceed a threshold maintained by evolutionarily conserved homeostatic mechanisms. How excess copper induces cell death, however, is unknown. Here, we show in human cells that copper-dependent, regulated cell death is distinct from known death mechanisms and is dependent on mitochondrial respiration. We show that copper-dependent death occurs by means of direct binding of copper to lipoylated components of the tricarboxylic acid (TCA) cycle. This results in lipoylated protein aggregation and subsequent iron-sulfur cluster protein loss, which leads to proteotoxic stress and ultimately cell death. These findings may explain the need for ancient copper homeostatic mechanisms.

Figure 1.. Copper ionophore-induced cell death is non-apoptotic, non-ferroptotic and non-necroptotic.

(A) PRISM Repurposing Secondary screen - growth-inhibition estimates for 1,448 drugs against 489 cell lines. (B) Viability of cells (MON) after treatment with elesclomol ± 10μM of indicated metals. (C) Viability of ABC1 cells was assessed at the indicated times after elesclomol-Cu (1:1 ratio) pulse treatment and growth in fresh media (D) Caspase 3/7 cleavage in ABC1 cells after 16 hours following indicted treatments (fold change over control). (E) G402 cells treated with the indicated concentrations of elesclomol or 25μM etoposide for 6 hours. (F) Viability of HMC18 cells or two HMC18 clones with Bax/Bak deleted after treatment with elesclomol-CuCl₂ (1:1) (top) and paclitaxel (bottom). (G) Heatmap of viability of cells pretreated overnight with 20μM necrostatin-1, 10μM ferrostatin-1, 1mM and 5mM N-Acetylcysteine, 30μM Z-VAD-FMK, 50μM D-Boc-FMK, 20μM TTM, 300μM L-NAME, 1μM Pepstatin A and 10μM DPQ and then treated with either 30nM elesclomol-CuCl₂ (1:1), 1μM ML162 (GPX4 inhibitor) or 40nM bortezomib for 72h (average of three replicas).(H) Schematic diagram of apoptosis, necroptosis, and ferroptosis. Inhibited pathways are marked in red. (B, D and F) mean ± SD, n≥3. (D,E) media supplemented with 1μM CuCl₂

Figure 2.. Mitochondria respiration regulates copper ionophore induced cell death.

(A) Viability of NCIH2030 cells grown in media containing either glucose or galactose treated with elesclomol-Cu (ratio 1:1). (B) Viability of ABC1 cells pretreated with indicated compounds (x-axis) and then treated with elesclomol (ES), NSC319726 or ML162 (y-axis). Average of at least three replicas is plotted and color coded. (C) Viability of ABC1 cells pretreated with 0.1μM rotenone (Rot), 0.1μM Antimycin A (AntiA) or 1μM FCCP and then treated with elesclomol. (D) Viability of ABC1 cells grown in control (21% O₂), hypoxia (1% O₂) or 50 μM FG-4592 (21% O₂) after treatment with elesclomol. (E) The oxygen consumption rate (OCR) was detected following treatment with 2.5 nM elesclomol (+1μM CuCl₂ in media) for 16 hours of ABC1 cells before (basal) and after the addition of oligomycin (ATP-linked), the uncoupler FCCP (maximal), or the electron transport inhibitor antimycin A/rotenone (baseline) (mean ± SD, n= 8). (F) Schematic of metabolites altered following elesclomol treatment of ABC1 cells (purple circles mark metabolites changing abundance as detailed in table S1. Larger circles –metabolites upregulated, smaller circles- metabolites downregulated). (A, C and D) mean ± SD, n≥ 3.

Figure 3.. FDX1 and lipoic acid genes are critical mediators of copper ionophore-induced cell death.

(A-C) Whole genome CRIPSR/Cas9 positive selection screen using two copper ionophores (Cu-DDC and elesclomol-copper) in OVISE cells (schematic on the left). Overlapping hits with FDR score< 0.01 were analyzed (right). Positive hits (resistance) are marked in blue, negative hits (sensitizers) in red (B-C) Summary scatter of the screen results for Cu-DDC (B) or elesclomol-copper (C). (D) Schematic of the lipoic acid (LA) pathway. Genes that scored in our genetic screens are marked as essential for copper-induced cell death. (E) Summary scatter indicating top hits in metabolism gene focused CRISPR–Cas9 gene knockout screen of A549 cells treated with 40nM of elesclomol-Cu(II) (1:1 ratio). Genes associated with the lipoic acid pathway are marked in blue, complex I related genes in orange and FDX1 in purple. (F) Viability of ABC1 cells with CRIPSR/Cas9 deletion of LIAS or FDX1 following treatment with elesclomol in the presence of 1μM CuCl₂ in the media. (G) Growth curve measurements of OVISE cells with CRIPSR/Cas9 deletion of LIAS and FDX1 in the presence of 20nM elesclomol. (F-G) mean ±SD of n≥3.

Figure 4.. FDX1 is an upstream regulator of protein lipoylation

(A) Correlation analysis of gene dependencies taken from the Achilles project; presented is the gene network that correlates with FDX1 deletion using FIREWORKS (51). The correlating genes are marked by their described functionality (lipoic acid pathway and mitochondria complex I and Fe-S cluster regulation). (B) A tissue microarray (TMA) of Non-small cell lung carcinoma (NSCLC) (n=57) were stained with LA and FDX IHC and expression was scored semi-quantitatively by two pathologists (S.C., S.S.), showing a strong direct correlation between LA and FDX expression (mean ± S.D.; p<0.0001). (C) Representative cases of NSCLC with correlated low (top-row) and high (bottom-row) expression of LA and FDX1 by IHC (scale bars 20μm). (D) Immunoblot of lipoylated proteins, FDX1, DLAT and actin from extracts of PSN1 cells with deletion of FDX1. (E) Basal oxygen consumption rate (OCR) as measured in PSN1 cells with CRIPSR/Cas9 deletion of FDX1, LIAS or AAVS1 genes (unpaired t-test *** p< 0.001). (F) Plot of the average Log2 fold change in metabolites between FDX1 KO and AAVS1 control K562 cell lines separated by functional annotations. Metabolites relevant to the lipoic acid pathway are marked in orange.

Figure 5.. Copper directly binds and promotes the oligomerization of lipoylated DLAT

(A) The binding of indicated proteins to copper (Cu), Cobalt (Co) and Nickel (Ni) was assessed by immunoblot analysis of eluted proteins from the indicated metal loaded resins. (B) Copper binding was assessed by loading cell lysates from either ABC1 AAVS1 or FDX1 KO cells on copper loaded resin followed by washing and analysis of the eluted proteins. Input and eluted proteins are presented. (C) Protein content was analyzed in A673 cells that were pulse (2hr) treated with the indicated concentrations of elesclomol. (D) Protein content was analyzed in ABC1 and A549 cells that were pulse treated with the indicated concentrations of elesclomol. (E-H) AAVS1 control or FDX1 KO ABC1 cells were pulse treated with indicated concentrations of elesclomol for 2 hours; protein oligomerization was analyzed after 24 hours by immunoblotting (E) and both wide-field (F) and confocal (G) immunofluorescence imaging (DLAT - green, Mitotracker- red, Hoechst -blue, phalloidin -white). (H) Foci were segmented and quantified in each condition(n=3, multiple unpaired t-test analysis was conducted with desired FDR(Q)= 1% *** q< 0.001). (C-H) 1μM CuCl₂ was supplemented to the media.

Figure 6.. Shared mechanisms in chemically and genetically induced copper dependent cell death.

(A) Proteomic analysis of control and elesclomol (100nM) pulse-treated ABC1 cells 24h post-treatment. Top (GO) enriched categories are presented (table S4). (B-C) Protein content in A673 (B) and AAVS1 and FDX1-KO ABC1 cells (C) 16h after 2h-pulse-treatment with elesclomol. (A-C) performed with 1μM CuCl₂ supplementation. (D) Copper homeostasis schematic. (E-F) 293T cells overexpressing GFP or SLC31A1 analyzed for viability 72h post-supplementation with CuCl₂ and (E) for protein content 24h post-treatment (F). (G) SLC31A1-overexpressing cells were pre-treated with 10μM necrostatin-1, 10μM ferrostatin-1, 100μM α-Tocopherol, 40μM Z-VAD-FMK, 10μM tetrathiomolybdate or 40μM Bathocuproinedisulfonic acid; Viability was measured after 72 hours of cells growing in the presence or absence of 3 μM CuCl₂. (H) Viability of ABC1 Ch2–2, FDX1, and LIAS-KO cells overexpressing SLC31A1 was analyzed 72h post-treatment with 2μM CuCl₂. In (G) and (H) viability is presented as box and whiskers plots representing interquartile ranges (boxes), medians (horizontal lines), and range (whiskers). For both, n≥5, and an ordinary one-way ANOVA with multiple comparisons (****adjusted p-value<0.0001). (I) Viability of control and 100μM BSO-pretreated A549 cells 48h post-treatment with indicated metals. Average of three replicas plotted. (J) Viability of control, FDX1 and LIAS-KO HEK293T cells pretreated with 20μM BSO and then with CuCl₂. (K) Protein content in Atpb7b−/− mouse livers (≥5 Atp7b−/−) compared to control (six Atp7b +/−; one wt). Multiple unpaired t-test analysis were conducted ***q< 0.001, **q< 0.01. (L) Schematic of mechanisms promoting copper-induced cell death. (E, J) mean ±SD of n≥4.