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A. Al-Zebeeby et al.
ty of chemoresistant K562 cells to A-1331852-mediated apoptosis, albeit to varying degrees (Figure 2C,D). While downregulation of SLC1A5 and GLS resulted in enhanced sensitivity to A-1331852-mediated apoptosis in the differ- ent cell lines tested, inhibition of other enzymes in the glutamine metabolic pathway produced more modest effects (Figure 2C,D and Online Supplementary Figure S7).
Targeting reductive carboxylation enhances sensitivity to BH3 mimetics
Metabolic supplementation of the glutamine-deprived cells with either glutamine or a-KG restored the resist- ance of K562 cells to A1331852-induced apoptosis (Figure 3A). Since glutamine-derived a-KG feeds into the tricar- boxylic acid cycle, we explored the functions of this cycle and its intermediates in chemoresistance to BH3 mimet- ics. For this, we supplemented glutamine-deprived cells with tricarboxylic acid intermediates, such as oxaloac- etate and citrate. Strikingly, supplementation with citrate, but not oxaloacetate, restored the resistance of K562 cells to A-1331852-induced apoptosis (Figure 3A). These results suggest that conversion of a-KG to citrate via reductive carboxylation may play a role in regulating sen- sitivity to BH3 mimetics.
AB
CD
Figure 2. Inhibition of glutamine uptake and metabolism enhances sensitivity to BH3 mimetics. (A) Deprivation of glutamine (Gln) for 16 h restores the apoptotic sensitivity of resistant [E] MAVER-1, K562 and H929 cells to the indicated BH3 mimetic for 4 h. (B) Scheme representing the pathway of glutamine uptake and metab- olism. (C) Apoptotic sensitivity of K562 resistant [E] cells exposed to A-1331852 (10 nM) for 4 h was restored following genetic knockdown for 72 h with the indicated short interfering (si) RNA. (D) Apoptotic sensitivity of K562 resistant [E] cells exposed to A-1331852 (10 nM) for 4 h was restored following pharmacological inhibition of glutamine uptake or metabolism with GPNA (5 mM) for 48 h, CB-839 (10 mM) for 72 h, azaserine (25 mM) for 16 h and AOA (500 mM) for 24 h but not with EGCG (50 mM) for 24 h. Western blots confirmed the knockdown efficiency of the different siRNA. ***P⩽0.001, **P⩽0.01. Error bars = mean ± standard error of mean (n=3). PS: phosphatidylserine; DMSO: dimethylsulfoxide.
Reductive carboxylation involves the conversion of a- KG to isocitrate (catalyzed by isocitrate dehydrogenases
1 and 2; IDH1/2), which then generates citrate (catalyzed by aconitase) (Figure 3B).23 While IDH1/2 catalyze reduc- tive carboxylation of a-KG, another isoform of isocitrate dehydrogenase, IDH3, catalyzes the reverse conversion of isocitrate to a-KG.23 Silencing the expression of IDH2 and aconitase, but not IDH3, restored the sensitivity of chemoresistant K562 cells to A-1331852-mediated apop- tosis (Figure 3C,D), suggesting that the availability of cit- rate could be associated with the chemoresistance pheno- type. To test this, IDH2-downregulated K562 cells were supplemented with citrate to identify whether addition of citrate could overcome the inhibition of reductive car- boxylation and revert the associated increase in A- 1331852-induced apoptosis. Supplementation with cit- rate, but not glutamine or a-KG, did indeed restore the chemoresistance of IDH2-downregulated cells (Figure 3E), thus confirming the involvement of reductive car- boxylation and the availability of citrate as crucial players in the observed chemoresistance.
Downregulation of lipogenesis and cholesterogenesis enhances sensitivity to BH3 mimetics
Since citrate generated as a consequence of reductive carboxylation of a-KG is a major source of carbon for lipid synthesis, we investigated whether inhibition of lipogenesis could enhance sensitivity to BH3 mimetics
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haematologica | 2019; 104(5)