ATG7 regulates energy metabolism, differentiation and survival of Philadelphia-chromosome-positive cells

Abstract

A major drawback of tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) is that primitive CML cells are able to survive TKI-mediated BCR-ABL inhibition, leading to disease persistence in patients. Investigation of strategies aiming to inhibit alternative survival pathways in CML is therefore critical. We have previously shown that a nonspecific pharmacological inhibition of autophagy potentiates TKI-induced death in Philadelphia chromosome-positive cells. Here we provide further understanding of how specific and pharmacological autophagy inhibition affects nonmitochondrial and mitochondrial energy metabolism and reactive oxygen species (ROS)-mediated differentiation of CML cells and highlight ATG7 (a critical component of the LC3 conjugation system) as a potential specific therapeutic target. By combining extra- and intracellular steady state metabolite measurements by liquid chromatography-mass spectrometry with metabolic flux assays using labeled glucose and functional assays, we demonstrate that knockdown of ATG7 results in decreased glycolysis and increased flux of labeled carbons through the mitochondrial tricarboxylic acid cycle. This leads to increased oxidative phosphorylation and mitochondrial ROS accumulation. Furthermore, following ROS accumulation, CML cells, including primary CML CD34(+) progenitor cells, differentiate toward the erythroid lineage. Finally, ATG7 knockdown sensitizes CML progenitor cells to TKI-induced death, without affecting survival of normal cells, suggesting that specific inhibitors of ATG7 in combination with TKI would provide a novel therapeutic approach for CML patients exhibiting persistent disease.

Keywords: ATG7; autophagy; chronic myeloid leukemia; energy metabolism; erythroid differentiation; glycolysis; oxidative phosphorylation; reactive oxygen species; tyrosine kinase inhibitor.

Figure 1.

ATG7 knockdown affects extracellular metabolite levels. (A) A schematic diagram of energy metabolism in mammalian cells: During glycolysis, glucose (that contains 6 carbons) is converted into 2 molecules of the 3-carbon metabolite pyruvate via a series of intermediate metabolites. Pyruvate can then either be used to produce lactate (3-carbon molecule which is often secreted from cells) or transferred to the mitochondria for further breakdown in the TCA cycle. The TCA cycle is a series of chemical reactions, which leads to generation of energy through the oxidation of acetate (in the form of acetyl coenzyme A; acetyl Co-A) into carbon dioxide (CO₂) and energy in the form of ATP, which is synthesized following OXPHOS in the mitochondrial ETC. U-¹³C₆ can be used to examine flow of labeled carbons (indicated by red circles) through these pathways. During the conversion of 3-carbon pyruvate to acetyl Co-A, one carbon is converted to CO₂ such that acetyl Co-A maintains 2 pyruvate-derived carbons (labeled) when it enters the TCA cycle. This can lead to build up of labeled carbons in TCA cycle intermediates when the pathway is activated. (B to H) shATG7-expressing K562 cells were cultured in normal medium (B to E) or in the presence of U-¹³C₆ (F to H). Extracellular glucose (B and F), lactate (C and G), glutamate (D and H) and glutamine (E) were measured in the medium by LC-MS following 24 h culture. Note, the significance levels for metabolites with variable labeled carbons, (i.e. Glu+1 => glutamate + 1 × ¹³C) are indicated in figure legends to the right of the graph: +1, etc. corresponds to glutamate containing 1X, etc. ¹³C. Two independent experiments were performed in triplicate. P values: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Figure 2.

Autophagy inhibition affects intracellular metabolite levels. (A to G) ShATG7-expressing K562 cells were cultured in the presence of U-¹³C₆ for 24 h. Following cell lysis, intercellular levels and incorporation of labeled carbons in glucose-6P (G6P) (A), P-enolpyruvate (PEP) (B), pyruvate (Pyr) (C), lactate (Lac) (D), citrate (Cit) (E), α-ketoglutarate (αKG) (F) and glutamate (Glu) (G) was measured by LC-MS. Two independent experiments were performed in triplicate. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. +2, etc. corresponds to metabolite containing 2X, etc. ¹³C.

Figure 3.

Autophagy inhibition reduces glycolysis and induces OXPHOS. ECAR (A) and OCR (B) were measured in shATG7-expressing cells using the Seahorse Extracellular Flux Analyzer. Glycolysis (relative to shCtrl-expressing cells) was calculated as “average ECAR following glucose addition minus average ECAR following inhibition of glycolysis using 2-DG.” Relative mitochondrial respiration was calculated as “basal OCR minus OCR following antimycin and rotenone (ETC inhibitors) treatment (legends for Fig. S3 provide more details). Three independent experiments were performed in quintuplicate. ECAR (C) and OCR (D) were measured in K562 cells following 24 h 10 µM HCQ treatments. Two independent experiments were performed in quintuplicate. (E and F) ShATG7-expressing K562 cells were cultured in the absence (E) or presence (F) of U-¹³C₆ for 24 h. Following cell lysis, intercellular levels and incorporation of labeled carbons in ATP was measured by LC-MS. Two independent experiments were performed in triplicate. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. +2, etc. corresponds to ATP containing 2X, etc. ¹³C.

Figure 4.

Elevated mitochondrial respiration induces ROS accumulation in CML cells. (A-B) levels of active mitochondria were measured in shATG7-expressing cells following staining of cells with TMRM (A) and MTR (B) using flow cytometry. (C) Colocalization of active mitochondria and lysosomes was measured in shATG7-expressing cells following staining of cells with MTR and LAMP1 using confocal microscopy. (D to F, as well as H and I) mitochondrial superoxide levels were measured by MitoSox staining in shATG7-expressing cells (D), K562 cells following 72 h 10 µM HCQ treatment alone and in combination with 10 nM NAC (E), CP CML CD34⁺ cells (n = 3) following 72 h 10 µM HCQ (F), K562 cells cultured in the presence of glucose (normal medium) or galactose for 72 h (H) and K562 cells treated with 2 and 4 mM oxamate for 72 h (I). Three independent experiments (A-F, H-I) were performed in duplicate. (G) OCR was measured in K562 cells cultured in the presence of 11 mM glucose or 11 nM galactose for 72 h. Three independent experiments were performed in quintuplicate. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Figure 5.

Enhanced OXPHOS drives differentiation of K562. (A to H) cellular profile was analyzed in K562 by flow cytometry for expression of TFRC (A, C, E, G and H) and GYPA (B, D and F) following 5 d culture in galactose (A and B), 5 d treatment with 2 µM and 4 µM oxamate (C and D), ATG7 knockdown alone (E and F) or +/− 10 nM NAC treatment (G) or 72 h 10 µM HCQ treatment +/− 10 nM NAC (H). Three independent experiments were performed in duplicate. (I and J) TFRC (I) and GYPA (J) levels were measured in CP CML CD34⁺ cells (n = 3) following 72 h 10 µM HCQ treatment. **, P ≤ 0.01; ***, P ≤ 0.001.

Figure 6.

ATG7 knockdown enhances the effects of TKI treatment on survival of CP CML CD34⁺ cells. Following ATG7 knockdown CP CML CD34⁺ (A to C, n=3) or non-CML CD34⁺ cells (D, n = 3) were sorted based on GFP expression. (A) The number of autophagy-related vesicles (filled double membrane vesicles, indicated with black arrows) was quantified using electron microscopy (total of 9 cells per arm). Representative pictures are shown. (B to D) The clonogenic ability of shCtrl or shATG7 CD34⁺ cells was evaluated by transferring cells to semisolid media following treatment with TKI for 3 d (B, D) or 6 d (C).