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Modulation of intermediary metabolism in cancer therapy
umented.1,14-19 This is a promising approach, as drugs tar- geting different stages of intermediary metabolism are already approved or in trials for treating different malig- nancies.20,21 In this study, we found a low level of resist- ance that developed in cells from patients with chronic lymphocytic leukemia (CLL) exposed to navitoclax. To mimic this modest resistance, we developed simple mod- els of resistance to different BH3 mimetics and demon- strated that downregulating glutamine uptake or metabo- lism as well as its downstream signaling cascades, such as reductive carboxylation, lipogenesis and cholesterogene- sis, result in enhanced apoptosis of cancer cells resistant to different BH3 mimetics, thus highlighting the possibil- ity that inhibition of key regulatory enzymes of these metabolic pathways may enhance sensitivity to BH3 mimetic therapy.
Methods
Reagents and antibodies
ABT-263, A-1331852 and A-1210477 were from AbbVie (North Chicago, IL, USA), ABT-199, epigallocatechin gallate (EGCG), CB-839, simvastatin, rapamycin and torin-1 from Selleck Chemicals (Houston, TX, USA), gamma-L-glutamyl-p- nitroanilide (GPNA) from Insight Biotechnology (Wembley, Middlesex, UK), azaserine from Cambridge Bioscience (Cambridge, UK), aminooxyacetate (AOA), sodium palmitate, dimethyl a-ketoglutarate, oxaloacetate and citrate from Sigma- Aldrich (Gillingham, UK), L-glutamine from Life Technologies (Paisley, UK) and GSK2194069, SB204990, atorvastatin, pitavas- tatin and bafilomycin A1 from Tocris (Abingdon, UK). Antibodies against PARP, BCL-2, MCL-1, BAX, BAK and GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), caspase-3, caspase-9, BCL-XL, BCL-w, BIM, PUMA, BAD, IDH2, ACL, ACO2, ATG5 and ATG7 from Cell Signaling Technology (MA, USA), BID from Prof. J. Borst (The Netherlands Cancer Institute, Amsterdam, the Netherlands), NOXA from Millipore (Watford, UK) and SLC1A5, GLS, GFAT, GLUD1, IDH3, FASN and HMGR from Abcam (Cambridge, UK).
Primary chronic lymphocytic leukemia cells and cell lines
Peripheral blood samples from CLL patients were obtained with the patients’ consent and ethics committee approval (06_Q2501_122) from Leicester Haematological Tissue Bank and cultured as described elsewhere.38 CLL samples were obtained from patients enrolled in a phase I/IIa study of ABT-263 (navito- clax) in patients with relapsed or refractory CLL (NCT00481091). Lymphocytes were purified and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Life Technologies Inc.). Alternatively, blood from patients was incubated at 37°C in 48-well plates and apoptosis assessed as described previously.39,40 Blood samples were collected prior to the first in vivo dose of navitoclax or 4 h after dosing during the lead-in period (day 1 of the lead-in period; L1D1), or day 1 of cycle 1 (C1D1), cycle 3 (C3D1) or cycle 5 (C5D1). Samples were collected 4 h after dosing as blood concentrations of ABT-263 were maximal at this time.41 For culture of CLL cells, mouse fibroblast L cells were irradiated with 75 Gy and seeded in 24- well plates (3 x105 cells/well). CLL cells were cultured at 1.5 x 106 cells/well on the L cells and removed when required by gentle washing with RPMI before treatment. Mantle cell lymphoma (MAVER-1), chronic myeloid leukemia (K562) and multiple myeloma (NCI-H929) cell lines were cultured in RPMI 1640
medium but the medium was supplemented with 0.02% 2-mer- captoethanol for culturing H929 cells. Cell lines were from either the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DMSZ; Braunshweig, Germany) or the American Type Culture Collection (ATCC; Middlesex, UK) and subjected to short tan- dem repeat profiling to confirm their identity.
Resistance models
The different resistance models to relevant BH3 mimetics were developed by treating control cells (represented as A in all schemes) of MAVER-1, K562 and H929 to the relevant BH3 mimetics, ABT-199 (10 nM), A-1331852 (10 nM) or A-1210477 (5 μM), respectively. In the first resistance model, cells were exposed to their appropriate BH3 mimetic for 24 h followed by 2 weeks without drug resulting in the cells depicted as B. These cells were then exposed to their appropriate BH3 mimetic for a further 24 h followed again by 2 weeks without drug resulting in C. This procedure was repeated twice more, resulting in E. In the second resistance model, cells were exposed to their appro- priate BH3 mimetic for 24 h followed by 8 weeks without drug, resulting in the cells depicted as A4. The cells were collected every 2 weeks and labeled as A1, A2 and A3, respectively. In the third resistance model, cells were exposed to increasing concen- trations of the appropriate BH3 mimetic every 5 days, resulting in cells depicted as A-a, A-b, A-c and A-d. The fourth model of resistance was made in a similar manner, but the 5-day treat- ment period was split into 2 days of treatment, followed by 3 days without drug, resulting in cells depicted as A-i, A-ii, A-iii and A-iv.
Metabolic deprivation, supplementation and apoptosis measurements
For glutamine deprivation experiments, cells were washed with phosphate-buffered saline and re-suspended in SILAC RPMI 1640 Flex Media (Life Technologies Inc.), supplemented with glucose (2 mg/mL) and 10% fetal bovine serum, for 16 h. For supplementation studies, the indicated concentrations of metabolites were added to the glutamine-free media, immedi- ately before glutamine deprivation. For lipid supplementation studies, sodium palmitate [dissolved in water at 70°C to form a stock concentration of 100 mM and added dropwise into fatty acid-free bovine serum albumin (10%) to produce a final con- centration of 10 mM] was supplemented in the culture media. The extent of apoptosis in cells following different treatments was quantified by fluorescence activated cell sorting (FACS) after having stained the cells with annexin V-fluorescein isothio- cyanate and propidium iodide to measure phosphatidylserine externalization, as previously described.42
Short interfering RNA knockdown, immunoprecipitation and western blotting
Cells were transfected with 10 nM of short interfering RNA (siRNA) against SLC1A5 (SI00079730), GLS (SI03155019), GFAT (SI03246355), GLUD1 (SI02654743), IDH2 (SI02654820), IDH3 (SI00300524), ACO2 (SI03019037), ACLY (SI02663332), FASN (SI00059752), HMGR (SI00017136), ATG5 (SI02633946) and ATG7 (SI04344830) from Qiagen Ltd. (Manchester, UK) using interferin (Polyplus Transfection Inc, NY, USA), according to the manufacturer's protocol and processed 72 h after transfection. Immunoprecipitation and western blotting were carried out according to standard protocols.43
Statistical analysis
One-way analysis of variance (ANOVA) multiple comparisons and the Fisher least significant difference test (P≤0.01) were per-
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