Inhibition of Folate Metabolism Drives Autophagy-Dependent Differentiation and Reduces Survival of Therapy-Resistant Leukaemic Stem Cells

Metabolic rewiring is an important hallmark of cancer. The folate metabolism pathway, also known as one-carbon (1C) metabolism, allows for transfer of 1C units through folate intermediates for biosynthetic processes, including precursors for DNA synthesis. Recent studies have shown that enzymes involved in the mitochondrial arm of 1C metabolism are overexpressed in a subset of aggressive cancers and that their expression affects responses to anti-metabolite drug treatments. However, the role of 1C metabolism in therapy resistant leukemic stem cells (LSCs) is currently unknown. Therefore, we aimed to investigate the activity and the impact of genetic and pharmacological inhibition of folate enzymes in primitive chronic myeloid leukaemia (CML) cells. We initially performed transcriptomic analysis of CD34+38- cells, from individuals with chronic phase CML (E-MTAB-2581). This revealed a significant upregulation of folate metabolism genes in CML LSCs, including serine hydroxymethyltransferase (SHMT2; p≤0.05), a key mitochondrial enzyme. To assess the activity of 1C metabolism in primitive cells we performed gas chromatography-mass spectrometry-mediated secretomic analysis using patient-derived, c-KIT enriched CML cells, which revealed a significant increase in the exchange rate of formate (folate intermediate necessary for purine synthesis) in CML cells, when compared to the secretome of normal counterparts (p<0.05). This reinforced the idea that 1C metabolism may be a metabolic dependency in CML. Following CRISPR-Cas9-mediated SHMT2 knockout (KO) in CML cell line, we observed a significant decrease in growth rate, together with a decrease in glycolytic capacity and oxygen consumption rate (p<0.01), suggesting impairment in proliferation and central carbon metabolism. Further metabolic characterisation of CML SHMT2 KO cells using liquid chromatography-mass spectrometry demonstrated a significant increase in AICAR, a purine biosynthesis intermediate and an AMP activated kinase (AMPK) activator. This prompted us to investigate the effect of 1C metabolism inhibition on AMPK. We found that AMPK phosphorylation on the conserved Thr 172 (a site that is phosphorylated under energy stress) was increased in SHMT2 KO cells, with similar effect seen following pharmacological inhibition of both SHMT2 and its cytosolic counterpart SHMT1 using SHIN1,which also promoted AMPK-dependent phosphorylation of the autophagy-inducing kinase ULK1 and downstream ULK1 target ATG13. Moreover, analysis of mitochondrial fraction revealed accumulation of mitochondrial fission related protein DRP1 and the mitophagy receptor NIX on mitochondria, hinting towards cellular interplay between 1C metabolism and mitochondrial homeostasis. Phenotypically, both pharmacological and genetic inhibition of SHMT1/2 induced the expression of erythropoiesis markers CD71 and Glycophorin A, which was reversed following formate supplementation. CRISPR-Cas9-mediated double AMPKα1/α2 KO revealed that the increased expression of these erythropoiesis markers following SHMT1/2 inhibition was independent of AMPK activity. Conversely, while NIX KO had no effect, pharmacological inhibition of ULK1 kinase activity, or genetic inhibition of ULK1 and ATG7 (protein important for autophagosome formation), prevented increased expression of CD71/Glycophorin A following SHMT1/2 inhibition. We next investigated the effect of 1C metabolism inhibition on differentiation and survival of primary CML cells. Of clinical relevance, pharmacological inhibition of SHMT1/2 promoted erythroid maturation of CD34+ CML cells (measured by expression of CD71, CD44, CD36 and Glycophorin A) when challenged with erythropoietin, which sensitises primitive cells to erythroid lineage commitment. Lastly, pharmacological inhibition of 1C metabolism decreased the colony formation capacity of CD34+ CML by 50%, with minimum effect on normal CD34+ cells. Moreover, combination treatment of SHIN1 with imatinib, a frontline treatment for CML patients, further increased the sensitivity of primary CML cells to imatinib by 40%. Overall, our novel findings indicate that disruption of the folate metabolism pathway inhibits central carbon metabolism in CML cells, promotes autophagy dependent, but AMPK independent maturation phenotype and has detrimental effect on the survival of primitive CML cells. Disclosures No relevant conflicts of interest to declare.

Evaluation of Apigenin Inhibiting Lactate Dehydrogenase Activity Based on CdTe Quantum Dots Fluorescence

Lactate dehydrogenase (LDH) is one of key enzymes in glucose metabolism pathway, which plays a critical role in cell metabolism. Inhibition of LDH can inhibit glycolysis process, thereby inhibiting the occurrence and development of tumor cells. Two kinds of LDH inhibitors, apigenin and emodin, were obtained by testing the IC50 of several natural products in LDH enzyme reaction. The IC50 of apigenin was about 1/3 of LDH inhibitor sodium oxalate. A new method to evaluate the performance of LDH inhibitors based on CdTe QDs was established at the same time, which provides a new idea for research on LDH enzyme inhibitors.

Inhibition of Caffeine Metabolism by Apiaceous and Rutaceae Families of Plant Products in Humans: In Vivo and In Vitro Studies

Daily consumption of caffeinated beverages is considered safe but serious health consequences do happen in some individuals. The Apiaceous and Rutaceae families of plant (ARFP) products are popular foods and medicines in the world. We previously reported significant amounts of furanocoumarin bioactive such as 8-methoxypsoralen, 5-methoxypsoralen, and isopimpinellin in ARFP products. As both caffeine and furanocoumarin bioactive are metabolized by the same hepatic CYP1A1/2 isozyme in humans, caffeine/ARFP product interactions may occur after co-administration. The objectives of the present study were to study in vivo loss of caffeine metabolizing activity by comparing the pharmacokinetics of caffeine in volunteers before and after pre-treatment with an ARFP extract, study the correlation between the decrease in hepatic CYP1A2 activity and the content of furanocoumarin bioactive in ARFP extracts, characterize CYP1A2 inactivation using in vitro incubations containing 14C-caffeine, a furanocoumarin bioactive, and human liver microsomes (HLMs), and provide a mechanistic explanation for both in vivo and in vitro data using the irreversible inhibition mechanism. The study results showed pre-treatment of volunteers with four ARFP extracts increased the area-under-the-concentration-time-curve (AUC0-inf) ratio of caffeine in the plasma ranging from 1.3 to 4.3-fold compared to the untreated volunteers indicating significant caffeine metabolism inhibition. The increases in AUC0-inf ratio also were linearly related to the effect-based doses of the furanocoumarins in the ARFP extracts, a finding which indicated caffeine metabolism inhibition was related to the content of furanocoumarin bioactive in an ARFP product. In vitro incubation studies also showed individual furanocoumarin bioactive were potent inhibitors of caffeine-N-demethylation; the IC50 for 8-methoxypsoralen 5-methoxypsoralen, and isopimpinellin were 0.09, 0.13, and 0.29 µM, respectively. In addition, CYP1A2 inactivation by individual furanocoumarin bioactive was concentration- and time-dependent involving the irreversible inhibition mechanism. The proposed irreversible inhibition mechanism was investigated further using 14C-labeled 8-methoxypsoralen and HLMs. The formation of 14C-adducts due to 14C-8-MOP-derived radioactivity bound to HLMs confirmed the irreversible inhibition of CYP1A2 activity. Thus, furanocoumarin bioactive metabolism in humans would result in reactive metabolite(s) formation inactivating CYP1A2 isozyme and inhibiting caffeine metabolism. Once the CYP1A2 isozyme was deactivated, the enzymic activity could only be regained by isozyme re-synthesis which took a long time. As a result, a single oral dose of ARFP extract administered to the human volunteers 3.0 h before still was able to inhibit caffeine metabolism.

Overexpression of Human Syndecan-1 Protects against the Diethylnitrosamine-Induced Hepatocarcinogenesis in Mice

Although syndecan-1 (SDC1) is known to be dysregulated in various cancer types, its implication in tumorigenesis is poorly understood. Its effect may be detrimental or protective depending on the type of cancer. Our previous data suggest that SDC1 is protective against hepatocarcinogenesis. To further verify this notion, human SDC1 transgenic (hSDC1+/+) mice were generated that expressed hSDC1 specifically in the liver under the control of the albumin promoter. Hepatocarcinogenesis was induced by a single dose of diethylnitrosamine (DEN) at an age of 15 days after birth, which resulted in tumors without cirrhosis in wild-type and hSDC1+/+ mice. At the experimental endpoint, livers were examined macroscopically and histologically, as well as by immunohistochemistry, Western blot, receptor tyrosine kinase array, phosphoprotein array, and proteomic analysis. Liver-specific overexpression of hSDC1 resulted in an approximately six month delay in tumor formation via the promotion of SDC1 shedding, downregulation of lipid metabolism, inhibition of the mTOR and the β-catenin pathways, and activation of the Foxo1 and p53 transcription factors that lead to the upregulation of the cell cycle inhibitors p21 and p27. Furthermore, both of them are implicated in the regulation of intermediary metabolism. Proteomic analysis showed enhanced lipid metabolism, activation of motor proteins, and loss of mitochondrial electron transport proteins as promoters of cancer in wild-type tumors, inhibited in the hSDC1+/+ livers. These complex mechanisms mimic the characteristics of nonalcoholic steatohepatitis (NASH) induced human liver cancer successfully delayed by syndecan-1.