Pathological glycogenesis through glycogen synthase 1 and suppression of excessive AMP kinase activity in myeloid leukemia cells

Abstract

The rapid proliferation of myeloid leukemia cells is highly dependent on increased glucose metabolism. Through an unbiased metabolomics analysis of leukemia cells, we found that the glycogenic precursor UDP-D-glucose is pervasively upregulated, despite low glycogen levels. Targeting the rate-limiting glycogen synthase 1 (GYS1) not only decreased glycolytic flux but also increased activation of the glycogen-responsive AMP kinase (AMPK), leading to significant growth suppression. Further, genetic and pharmacological hyper-activation of AMPK was sufficient to induce the changes observed with GYS1 targeting. Cancer genomics data also indicate that elevated levels of the glycogenic enzymes GYS1/2 or GBE1 (glycogen branching enzyme 1) are associated with poor survival in AML. These results suggest a novel mechanism whereby leukemic cells sustain aberrant proliferation by suppressing excess AMPK activity through elevated glycogenic flux and provide a therapeutic entry point for targeting leukemia cell metabolism.

Figure 1. Metabolomics analysis of human myeloid leukemia cells

(a) Unsupervised hierarchical clustering of KU812, Molm-13 and HEL metabolite profiles, comparing control treated cells (−) to tyrosine kinase inhibitor treated cells (+; 3.5 μM imatinib, 0.8 nM quizartinib and 400nM ruxolitinib, respectively) (p<0.05; change in metabolite levels >1.5-fold). Reduced metabolite levels are indicated by negative numbers (blue) in the heatmap. (b) Venn diagram obtained from comparison of metabolite profiles of KU812, Molm-13 and HEL in response to inhibitors of their respective oncogenic tyrosine kinases (left) or metabolite profiles of AML and CML patient specimens, compared to normal controls (n=3) (right). (c) Changes in metabolite levels consistently observed in myeloid leukemia cells, as indicated (p<0.05). (d) Changes in glycogen levels were measured in cellular extracts of KU812, MEG-01, K562, MOLM-13 and HEL cells in response to inhibitors of their respective oncogenic tyrosine kinases. *Significant differences (p<0.05) were observed between control and treated cells.

Figure 2. Dysregulation of the glycogen synthesis pathway in myeloid leukemia cells

(a) Schematic representation of GYS1, GYS2 (glycogen synthase 1 or 2) and GBE1 (1,4-alpha-glucan-branching enzyme 1) involved in the biochemical pathway for glycogen synthesis from UDP-D-glucose. (b) Elevated levels of GYS1, GYS2, or GBE1 were observed in AML patient specimens, as indicated (changes in expression >1.6-fold). (c) Survival distribution for AML patients with upregulation of GYS1, GYS2 or GBE1. The p-value denotes significant differences in survival between the query group and patients without overexpression of the genes within the query. (d) Changes in expression of genes involved in metabolism in response to imatinib (1μM; ◇) in KU812 cells were determined by real-time PCR relative to their respective p-values (volcano plot). Red symbols indicate changes in expression of GYS1, GYS2 and GBE1. The horizontal dashed line indicates the p=0.005 significance level and the vertical dotted lines indicate 2-fold changes.

Figure 3. Glycogen production is regulated at multiple levels

Expression of AMPKα, AMPKα (pT¹⁷²), GYS1, GYS1 (pS^8/11), GYS1 (pS⁶⁴¹) and β-actin was determined by immunoblotting in KU812 cells that were left untreated or treated for 18h with (a) imatinib (1 μM) or (b) resveratrol (10 μM) and metformin (1 mM). Changes in glycogen levels were measured in cellular extracts of (c) KU812 or KU812^IR cells in response to treatment (72h) with either metformin (1 mM), resveratrol (10 μM), AICAR (1mM) and A769662 (100 μM) (n=3) or (d) in cellular extracts of cell lines (K562, MOLM-13, HEL and OCI-AML3) as well as primary patient specimens in response to metformin (1 mM) and resveratrol (10 μM). *Significant differences (p<0.05) were observed between control and treated cells.

Figure 4. Glycogenesis is required for metabolic reprogramming and associated with decreased AMPK activation

(a) KU812 cells containing an active form of AMPKα2 (ΔAMPK) were compare to parental KU812 and relative changes in glycogen, cell growth and lactate were measured (left). The expression of HA-tagged ΔAMPK was determined by immunoblotting (right). (b) KU812 cells containing scrambled shRNA (Scr) or GYS1-targeting constructs (A, B or E) were used. Changes in glycogen, lactate, or ROS levels (left) and GYS1 protein expression (right) were measured. (c) KU812 cells expressing GYS1-targeting shRNA construct B were treated with glycogen phosphorylase inhibitor (1 μM) or left untreated. Glycogen levels and AMPK phosphorylation (ELISA) were measured, as indicated. *Significant differences (p<0.05) were observed between control and treated cells.

Figure 5. GYS1 is required for increased growth

Cell growth was measured in KU812 cells expressing Scr shRNA or GYS1-targeting shRNA construct B treated with different concentrations of (a) imatinib or (b) glycogen phosphorylase inhibitor. (c) KU812 cells containing scrambled shRNA (Scr) or GYS1-targeting constructs (A, B or E) were used to measure growth in the presence of 4.5g/L or 0.4 g/L glucose. (d) In vivo tumor formation of KU812 cells with targeted knockdown of GYS1 construct B (◇) or scrambled shRNA (□) was determined in SCID/Beige mice (n=8). The volumes of subcutaneous tumors were measured and compared for 23 days after initial injection, as indicted. *Significant differences (p<0.05) were observed between control and treated cells.