Caspase-10 inhibits ATP-citrate lyase-mediated metabolic and epigenetic reprogramming to suppress tumorigenesis

Caspase-10 belongs to the class of initiator caspases and is a close homolog of caspase-8. However, the lack of caspase-10 in mice and limited substrate repertoire restricts the understanding of its physiological functions. Here, we report that ATP-citrate lyase (ACLY) is a caspase-10 substrate. Caspase-10 cleaves ACLY at the conserved Asp1026 site under conditions of altered metabolic homeostasis. Cleavage of ACLY abrogates its enzymatic activity and suppresses the generation of acetyl-CoA, which is critical for lipogenesis and histone acetylation. Thus, caspase-10-mediated ACLY cleavage results in reduced intracellular lipid levels and represses GCN5-mediated histone H3 and H4 acetylation. Furthermore, decline in GCN5 activity alters the epigenetic profile, resulting in downregulation of proliferative and metastatic genes. Thus caspase-10 suppresses ACLY-promoted malignant phenotype. These findings expand the substrate repertoire of caspase-10 and highlight its pivotal role in inhibiting tumorigenesis through metabolic and epigenetic mechanisms.

subjected to serum starvation for the indicated time points. The cells were then harvested and western blotting was performed. c A549 control (A549) and A549 p53 knockdown (A549 p53kd) cells were subjected to metformin (5mM) treatment for the indicated time points. The cells were then harvested and relative mRNA levels were analyzed. Error bars are means ± SD of three biological replicates. Statistical analyses were done using two-way ANOVA (Tukey's post hoc test). **P<0.01. d A549 control and A549 p53 knockdown (A549 p53kd) cells were subjected to metformin (5mM) treatment for the indicated time points. The cells were then harvested and western blotting was performed. e HCT116 control (HCT116) and p53 knockdown (HCT116 p53kd) cells were subjected to glucose starvation for indicated time points.
The cells were harvested and western blotting was performed. f IMR90 control (IMR90) and p53 knockdown (IMR90 p53kd) cells were subjected to glucose starvation for indicated time points.
The cells were harvested and western blotting was performed. g A549 cells were subjected to glucose starvation for indicated time points. The cells were then harvested and TUNEL assay was performed. Error bars are means ± SD of three biological replicates. Statistical analyses were done using unpaired t-test. ***P<0.001. h A549 control (control) and caspase-3 knockdown (CASP3kd) cells were harvested and western blotting was performed for the indicated proteins (top panel). A549 control (control) and caspase-3 knockdown (CASP3kd) cells were subjected to unstressed (25mM) or glucose starvation conditions (5mM) for 36 hours.
The cells were then harvested and caspase-10 activity was examined (bottom panel). Error bars are means ± SD of three biological replicates. Statistical analyses were done using two-way ANOVA (Bonferroni's post hoc test). **P<0.01.
Supplementary Figure 2. ACLY is a caspase-10 substrate a His-CASP10 mut was bacterially expressed and purified. The indicated proteins were run on SDS-PAGE (top panel). In vitro binding assay was performed followed by western blotting (bottom panel). b In vitro ACLY cleavage assay was performed. c A549 control and CASP10kd cells were subjected to serum starvation for indicated time points. A549 CASP10kd cells were infected with Ad-CASP10 or Ad-CASP10 mut during the last 12 hours prior to the end of the 48 hour time point. The cells were harvested and western blotting was performed. d HCT116 control and CASP10kd cells were subjected to glucose starvation for indicated time points. HCT116 CASP10kd cells were infected with Ad-CASP10 or Ad-CASP10 mut during the last 12 hours prior to the end of the 48 hour time point. The cells were harvested and western blotting was performed. e IMR90 control and CASP10kd cells were subjected to glucose starvation for indicated time points. IMR90 CASP10kd cells were infected with Ad-CASP10 or Ad-CASP10 mut during the last 12 hours prior to the end of the 48 hour time point. The cells were harvested and western blotting was performed. f A549 ACLYkd cells were co-transfected with HA-tagged caspase-10, and empty vector, FLAG-tagged wild-type (ACLY) or mutant ACLY (ACLY D1026A ), and treated with caspase-10 inhibitor (Q-AEVD-FMK) (25μM). 24 hours post-transfection, the cells were harvested and subjected to immunoprecipitation. g A549 ACLYkd cells were stably transfected with wild-type (ACLY) or mutant ACLY (ACLY D1026A ). These cells were subjected to serum starvation for indicated time points followed by western blotting. h A549 ACLYkd cells were stably transfected with wild-type (ACLY) or mutant ACLY (ACLY D1026A ). These cells were then subjected to metformin treatment for indicated time points followed by western blotting. i HCT116 ACLYkd cells were stably transfected with empty vector, wild-type (ACLY) or mutant ACLY (ACLY D1026A ). These cells were subjected to glucose starvation for indicated time points followed by western blotting. j IMR90 ACLYkd cells were stably transfected with empty vector, wild-type (ACLY) or mutant ACLY (ACLY D1026A ). These cells were subjected to glucose starvation for indicated time points followed by western blotting.
Supplementary Figure 3. Caspase-10 plays a key role in maintenance of acetyl-CoA levels a A549 control and A549 CASP10kd cells were subjected to serum starvation for indicated time points. ACLY activity was examined. Error bars are means ± SD of three biological replicates.
Statistical analyses were done using two-way ANOVA (Bonferroni's post hoc test). *P<0.05, **P<0.01. b A549 control and A549 CASP10kd cells were subjected to metformin treatment for indicated time points. ACLY activity was examined. Error bars are means ± SD of three biological replicates. Statistical analyses were done using two-way ANOVA (Bonferroni's post hoc test). **P<0.01. c HCT116 control and HCT116 CASP10kd cells were subjected to glucose starvation for indicated time points. ACLY activity was examined. Error bars are means ± SD of three biological replicates. Statistical analyses were done using two-way ANOVA (Bonferroni's post hoc test). **P<0.01. d IMR90 control and IMR90 CASP10kd cells were subjected to glucose starvation for indicated time points. ACLY activity was examined. Error bars are means ± SD of three biological replicates. Statistical analyses were done using twoway ANOVA (Bonferroni's post hoc test). *P<0.05. e A549 control and A549 CASP10kd cells were subjected to serum starvation for indicated time points. Acetyl-CoA levels were quantified.