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Modulation of intermediary metabolism in cancer therapy
(Figure 4A). Using a complementary approach of genetic and pharmacological inhibition of ATP-citrate lyase (ACLY), which catalyzes the conversion of citrate to acetyl-CoA,24,25 as well as fatty acid synthase (FASN), which synthesizes long chain fatty acids following the condensation of acetyl-CoA and malonyl-CoA,26,27 we identified that modulation of the lipogenesis pathway, using either genetic silencing or pharmacological inhibi- tion of ACLY (with SB204990) or FASN (with GSK2194069) could enhance sensitivity of cells to BH3 mimetics (Figure 4B,C and Online Supplementary Figure S7C,D). Furthermore, metabolic supplementation with palmitate (the product of FASN (Figure 4A) in cells treated with GSK2194069 reverted the sensitized cells to their original chemoresistant phenotype (Figure 4D), thus obvi- ating a requirement for FASN. These findings conclusive- ly demonstrated that enhanced lipogenesis was associat- ed with chemoresistance to BH3 mimetics and targeting lipogenesis could circumvent such resistance by enhanc- ing BH3 mimetic-mediated apoptosis.
Acetyl-CoA generated from citrate can also feed into the cholesterol biosynthetic pathway, thus resulting in enhanced cholesterol production in cells. Targeting the rate-limiting step of cholesterol biosynthesis (catalyzed
by HMG-CoA reductase; HMGR), either by genetic knockdown (Figure 4E) or pharmacological inhibition, using three widely used statins, simvastatin, atorvastatin and pitavastatin (Figure 4F), reversed resistance and restored the sensitivity of cells to BH3 mimetics (Figure 4E,F and Online Supplementary Figure S7E). Taken together, these data demonstrate that inhibition of several key players in lipid synthesis, including ACLY, FASN and HMGR, enhances the sensitivity to BH3 mimetics.
Targeting the mammalian target of rapamycin signaling cascade enhances sensitivity to BH3 mimetics
Since glutamine metabolism has been extensively implicated in mammalian target of rapamycin (mTOR) signaling,22,28 we speculated whether targeting mTOR kinases could enhance sensitivity to BH3 mimetics. Inhibition of mTOR kinases with rapamycin and torin-1 resulted in significant sensitization of cells to BH3-medi- ated apoptosis (Figure 5A and Online Supplementary Figure S7F). To identify whether torin-1-mediated sensitization of cells to apoptosis was due to autophagy, we exposed the sensitive and resistant cells to bafilomycin A1 (Baf A1), which blocks autophagic flux by preventing lysoso- mal fusion of the autophagosomes. Exposure to
ABC
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Figure 3. Modulation of reductive carboxylation enhances sensitivity to BH3 mimetics. (A) K562 sensitive [A] and resistant [E] cells were cultured in normal RPMI medium or glutamine-free medium with and without the supplementation of glutamine (2 mM), exposed to A-1331852 (10 nM) for 4 h and the extent of apoptosis assessed. Addition of citrate (4 mM) and α-ketoglutarate (a-KG) (4 mM) but not oxaloacetate (4 mM) for 16 h reversed the sensitivity of the resistant [E] cells in glu- tamine-deprived media. (B) Scheme representing the link between the tricarboxylic acid (TCA) cycle and reductive carboxylation. (C) K562 sensitive [A] and resistant [E] cells were transfected with short interfering (si) RNA against IDH2, IDH3 and aconitase for 72 h, followed by exposure for 4 h to A-1331852 and then apoptosis was assessed. (D) Western blots confirmed the knockdown efficiency of the different siRNA. (E) K562 [A] and [E] cells, transfected with a siRNA against IDH2 for 72 h, were glutamine-deprived and then given or not supplementation with glutamine (2 mM), a-ketoglutarate or citrate (both at 4 mM) for 16 h and the extent of apop- tosis following exposure to A-1331852 (10 nM) for 4 h was assessed. ***P⩽0.001. Error bars = mean ± standard error of mean (n=3). PS: phosphatidylserine; DMSO: dimethylsulfoxide.
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