Targeting intermediary metabolism enhances the efficacy of BH3 mimetic therapy in hematologic malignancies

Abstract

BH3 mimetics are novel targeted drugs with remarkable specificity, potency and enormous potential to improve cancer therapy. However, acquired resistance is an emerging problem. We report the rapid development of resistance in chronic lymphocytic leukemia cells isolated from patients exposed to increasing doses of navitoclax (ABT-263), a BH3 mimetic. To mimic such rapid development of chemoresistance, we developed simple resistance models to three different BH3 mimetics, targeting BCL-2 (ABT-199), BCL-X_L (A-1331852) or MCL-1 (A-1210477), in relevant hematologic cancer cell lines. In these models, resistance could not be attributed to either consistent changes in expression levels of the anti-apoptotic proteins or interactions among different pro- and anti-apoptotic BCL-2 family members. Using genetic silencing, pharmacological inhibition and metabolic supplementation, we found that targeting glutamine uptake and its downstream signaling pathways, namely glutaminolysis, reductive carboxylation, lipogenesis, cholesterogenesis and mammalian target of rapamycin signaling resulted in marked sensitization of the chemoresistant cells to BH3 mimetic-mediated apoptosis. Furthermore, our findings highlight the possibility of repurposing widely used drugs, such as statins, to target intermediary metabolism and improve the efficacy of BH3 mimetic therapy.

Figure 1.

Hematologic malignancies rapidly acquire resistance to BH3 mimetics. (A) Blood samples collected from patients with chronic lymphocytic leukemia (CLL) (n=5), either prior to the first in vivo dose of navitoclax or 4 h after dosing during different stages of treatment - day 1 of the initial lead-in-period (L1D1), day 1 of cycle 1 (C1D1), day 1 of cycle 3 (C3D1) or day 1 of cycle 5 (C5D1) - were incubated ex vivo and the extent of apoptosis in the CD19⁺ CLL cells was assessed at the indicated time points by measuring phosphatidylserine (PS) externalization. (B) CLL cells isolated from these patients at the beginning of each treatment cycle, as indicated in the figure, were exposed in vitro to increasing concentrations of ABT-263 and the extent of apoptosis was assessed: half maximal inhibitory concentration (IC₅₀) values are shown. (C) Scheme for establishing resistance to specific BH3 mimetics in relevant hematologic cell lines, as explained in the Methods section. Sensitive [A] and resistant [E] cells of MAVER-1, K562 and H929 cell lines were exposed for 4 h to ABT-199 (10 nM), A-1331852 (10 nM) and A-1210477 (5 μM), respectively, and apoptosis was assessed. (D-F) Combinations with some but not all BH3 mimetics restored apoptotic sensitivity of resistant [E] MAVER-1, K562 and H929 cells exposed for 4 h to ABT-199 (10 nM), A-1331852 (10 nM) or A-1210477 (5 μM), respectively. ***P⩽0.001, **P⩽0.01. Error bars = mean ± standard error of mean (n=3).

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 metabolism. (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 μM) for 72 h, azaserine (25 μM) for 16 h and AOA (500 μM) for 24 h but not with EGCG (50 μM) 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.

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 (α-KG) (4 mM) but not oxaloacetate (4 mM) for 16 h reversed the sensitivity of the resistant [E] cells in glutamine-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), α-ketoglutarate or citrate (both at 4 mM) for 16 h and the extent of apoptosis 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.

Figure 4.

Inhibition of lipogenesis and cholesterogenesis enhances sensitivity to BH3 mimetics. (A) Scheme representing reductive carboxylation, lipogenesis and cholesterogenesis. (B) Apoptotic sensitivity of K562 resistant [E] cells exposed to A-1331852 (10 nM) for 4 h was restored following genetic knockdown for 72 h of key enzymes in fatty acid synthesis. Western blots confirmed the knockdown efficiency of the different short interfering (si) RNA. (C) Apoptotic sensitivity of K562 resistant [E] cells exposed to A-1331852 (10 nM) for 4 h was restored following pharmacological inhibition of key enzymes in fatty acid synthesis using SB204990 (1 μM) for 72 h or GSK2194069 (100 nM) for 48 h. (D) Metabolic supplementation of K562 sensitive [A] and resistant [E] cells with palmitate (50 μM) for 48 h prior to the exposure of cells to GSK2194069 (100 nM) overcame the sensitizing effect of GSK2194069 on A-1331852-mediated apoptosis. (E) Genetic knockdown for 72 h of HMGR or (F) pharmacological inhibition of HMGR by simvastatin (250 nM) for 72 h, atorvastatin (10 μM) for 48 h or pitavastatin (1 μM) for 72 h. ***P⩽0.001; **P⩽0.01. Error bars = mean ± standard error of mean (n=3). PS: phosphatidylserine; DMSO: dimethylsulfoxide.

Figure 5.

Modulation of mammalian target of rapamycin signaling enhances sensitivity to BH3 mimetics independently of autophagy. (A) Apoptotic sensitivity of K562 resistant [E] cells exposed to A-1331852 (10 nM) for 4 h was restored following pharmacological inhibition of mTOR signaling using rapamycin (100 nM) or torin-1 (10 nM) for 16 h. (B) Inhibition of mTOR-regulated autophagy using 3-MA (10 mM) or bafilomycin A1 (100 nM) for 1 h, followed by torin-1 (10 nM) for a further 16 h, resulted in varying effects on A-1331852-mediated apoptosis. (C) Genetic knockdown of ATG5 and ATG7 for 72 h failed to revert torin-1 (10 nM)-mediated sensitization of apoptosis in K562 resistant [E] cells, following A-1331852 (10 nM) for 4 h. Western blots confirmed the knockdown efficiency of ATG5 and ATG7 short interfering (si) RNA. ***P⩽0.001. Error bars = mean ± standard error of mean (n=3). (D) Scheme representing glutamine uptake by SLC1A5 (inhibited by GPNA), glutaminolysis (inhibited by CB-839) to generate α-ketoglutarate, reductive carboxylation of α-ketoglutarate to generate citrate, which produces acetyl-CoA by a reaction catalyzed by ACLY (inhibited by SB204990), which eventually results in lipogenesis (inhibited by GSK2194069) and cholesterogenesis (inhibited by statins). Glutamine uptake, metabolism and its downstream signaling cascade can feed into mTOR signaling (inhibited by torin-1), all of which promote cell growth. In this study, we demonstrate that modulation of these distinct intermediary metabolic pathways could successfully sensitize cancer cells to BH3 mimetic-mediated apoptosis. PS: phosphatidylserine; DMSO: dimethylsulfoxide; TCA: tricarboxylic acid.

Figure 6.

Inhibition of glutaminase and HMG-CoA reductase circumvents resistance to navitoclax-mediated apoptosis in primary chronic lymphocytic leukemia cells. Chronic lymphocytic leukemia cells isolated from five patients during the initial lead-in-period (L1D1) or day 1 of cycle 5 (C5D1) were cultured ex vivo on a feeder layer for 24 h and then exposed for a further 24 h to (A) CB-839 (50 nM) or (B) simvastatin (10 nM), and removed from the feeder layer for further exposure to navitoclax (50 nM) for 4 h. The extent of apoptosis was assessed as before. *P⩽0.05. Error bars = mean ± standard error of mean (n=5). PS: phosphatidylserine; DMSO: dimethylsulfoxide.