Abstract
Small molecule kinase inhibitors have dramatically changed treatment of patients with myeloid malignancies, in particular chronic myeloid leukemia (CML), myeloproliferative neoplasms and some acute myeloid leukemias (AML). Nevertheless, genomic instability is frequently observed, resulting in the development of mutations that confer drug resistance. Our previous data suggest that myeloid malignancies are associated with metabolic reprogramming, allowing transformed cells to cover their enhanced energy needs and to provide biomolecules for anabolic processes. In patient-derived KU812 (BCR-ABL+), HEL (JAK2.V617F+) and Molm13 (FLT3-ITD+) cells, the transforming tyrosine kinase activity was observed to be associated not only with an increased glycolytic flux and a dependency on glucose for cell growth and viability, but also with a significant increase in glycogen production. In particular, the expression of three key enzymes within the glycogen synthase pathway, including UGP2 (UDP-glucose pyrophosphorylase 2), GYS1 (glycogen synthase 1) and GBE1 (glycogen branching enzyme 1), was found to be increased. We detected only trace amounts of the liver-specific GYS2. However, optimal glycogen production was found to be strictly dependent on the presence of glucose and required expression of the rate-limiting enzyme, GYS1. Surprisingly, targeted knockdown of GYS1 not only reduced glycogen levels, but it also inhibited growth, lowered lactate production and mitochondrial metabolism. GYS1 can be regulated in part through inhibitory serine phosphorylation by glycogen synthase kinases 3α or 3β (GSK3) at S641, downstream of AKT and PIM1/2 kinases, or AMP kinase (AMPK) at S8, downstream of the LKB1 kinase, although other kinases may also play a role in GYS1 phosphorylation. Interestingly, a truncated and constitutively active form of AMPK reduced glycogen levels, and inhibited metabolic reprogramming and cell growth in KU812 cells. Further, activators of AMPK, such as the anti-diabetic drugs resveratrol and metformin, mimic the loss of GYS1 in myeloid leukemia cells. Using a semi-quantitative mass spectrometry method (LC-MS/MS), we analyzed key metabolomic changes in cells in response to GYS1 knockdown and contrasted them to cells treated with these drugs. The results show a significant overlap in key metabolic pathways, in particular a reduction in early glycolytic markers (glucose-6-phospate, fructose-6-phosphate) and citric acid cycle intermediates (malate, fumarate). Additional reduction in the levels of essential amino acids was observed as well. To further test the efficacy of AMPK activation, we transfected the KU812 cells with an imatinib-resistant form of BCR-ABL containing the T315I mutation. While these cells were resistant to imatinib, as expected, both resveratrol and metformin efficiently inhibited growth and glycogen production in T315I-expressing cells, similar to levels in wild-type KU812 cells. Small molecule drugs that directly target GYS1 or activate AMPK would be expected to specifically inhibit glycogen production and to have activity in drug-resistant myeloid leukemias and other malignancies with elevated glycogen levels.
Disclosures:
No relevant conflicts of interest to declare.
Pathological glycogenesis through glycogen synthase 1 and suppression of excessive AMP kinase activity in myeloid leukemia cells