RNAi screening reveals a synthetic chemical-genetic interaction between ATP synthase and PFK1 in cancer cells.

To meet cellular bioenergetic and biosynthetic demands, cancer cells remodel their metabolism to increase glycolytic flux, a phenomenon known as the Warburg effect and believed to contribute to cancer malignancy. Among glycolytic enzymes, phosphofructokinase-1 (PFK1) has been shown to act as a rate-limiting enzyme and to facilitate the Warburg effect in cancer cells. In this study, however, we found that decreased PFK1 activity did not affect cell survival or proliferation in cancer cells. This raised a question regarding the importance of PFK1 in malignancy. To gain insights into the role of PFK1 in cancer metabolism and the possibility of adopting it as a novel anticancer therapeutic target, we screened for genes that caused lethality when they were knocked down in the presence of tryptolinamide (TLAM), a PFK1 inhibitor. The screen revealed a synthetic chemical-genetic interaction between genes encoding subunits of ATP synthase (complex V) and TLAM. Indeed, after TLAM treatment, the sensitivity of HeLa cells to oligomycin A (OMA), an ATP synthase inhibitor, was 13,000 times higher than that of untreated cells. Furthermore, this sensitivity potentiation by TLAM treatment was recapitulated by genetic mutations of PFK1. By contrast, TLAM did not potentiate the sensitivity of normal fibroblast cell lines to OMA, possibly due to their reduced energy demands compared to cancer cells. We also showed that the PFK1-mediated glycolytic pathway can act as an energy reservoir. Selective potentiation of the efficacy of ATP synthase inhibitors by PFK1 inhibition may serve as a foundation for novel anticancer therapeutic strategies.

Chemical reversal of abnormalities in cells carrying mitochondrial DNA mutations.

Mitochondrial DNA (mtDNA) mutations are the major cause of mitochondrial diseases. Cells harboring disease-related mtDNA mutations exhibit various phenotypic abnormalities, such as reduced respiration and elevated lactic acid production. Induced pluripotent stem cell (iPSC) lines derived from patients with mitochondrial disease, with high proportions of mutated mtDNA, exhibit defects in maturation into neurons or cardiomyocytes. In this study, we have discovered a small-molecule compound, which we name tryptolinamide (TLAM), that activates mitochondrial respiration in cybrids generated from patient-derived mitochondria and fibroblasts from patient-derived iPSCs. We found that TLAM inhibits phosphofructokinase-1 (PFK1), which in turn activates AMPK-mediated fatty-acid oxidation to promote oxidative phosphorylation, and redirects carbon flow from glycolysis toward the pentose phosphate pathway to reinforce anti-oxidative potential. Finally, we found that TLAM rescued the defect in neuronal differentiation of iPSCs carrying a high ratio of mutant mtDNA, suggesting that PFK1 represents a potential therapeutic target for mitochondrial diseases.

A novel GSK3 inhibitor that promotes self-renewal in mouse embryonic stem cells.

Small molecules that regulate cell stemness have the potential to make a major contribution to regenerative medicine. In the course of screening for small molecules that affect stemness in mouse embryonic stem cells (mESCs), we discovered that NPD13432, an aurone derivative, promoted self-renewal of mESCs. Normally, mESCs start to differentiate upon withdrawal of 2i/LIF. However, cells treated with the compound continued to express endogenous Nanog, a pluripotency marker protein essential for sustaining the undifferentiated state, even in the absence of 2i/LIF. Biochemical characterization revealed that NPD13432 inhibited GSK3α and GSK3ß with IC50 values of 92 nM and 310 nM, respectively, suggesting that the compound promotes self-renewal in mESCs by inhibiting GSK3. The chemical structure of the compound is unique among known molecules with this activity, providing an opportunity to develop new inhibitors of GSK3, as well as chemical tools for investigating cell stemness.

Identification of the determinants of 2-deoxyglucose sensitivity in cancer cells by shRNA library screening.

Combining glycolytic inhibition with other anti-cancer therapies is a potential approach to treating cancer. In this context, we attempted to identify genes that determine sensitivity to 2-deoxyglucose (2DG), a glycolytic inhibitor, in cancer cells using pooled shRNA libraries targeting ∼15,000 genes. The screen revealed that COPB1 and ARCN1, which are essential in retrograde transport, as determinants of sensitivity to 2DG: silencing of COPB1 or ARCN1 expression sensitized cells to 2DG toxicity. To address the mechanism of potentiation of 2DG toxicity by inhibition of COPI-mediated transport, we focused on the role of lipolysis as an alternate source of energy upon inhibition of glycolysis. In the process of lipolysis, COPI-mediated transport is required for localization to lipid droplets of adipose triglyceride lipase (ATGL), a key enzyme that produces fatty acids from triacylglycerol as a substrate for ß-oxidation. The ATGL inhibitor atglistatin potentiated 2DG toxicity, consistent with a model in which a defect in COPI-mediated transport of ATGL to lipid droplets inhibits energy supply, thereby sensitizing cells to glycolytic inhibition. Collectively, our data demonstrated that a defect in COPI-mediated transport or pharmacological inhibition of ATGL potentiates 2DG toxicity in cancer cells, possibly due to a reduction in the energy supply.

Isoquercitrin from Apocynum venetum L. produces an anti-obesity effect on obese mice by targeting C-1-tetrahydrofolate synthase, carbonyl reductase, and glutathione S-transferase P and modification of the AMPK/SREBP-1c/FAS/CD36 signaling pathway in mice in vivo.

In the present study, mice with high-fat-diet-induced obesity were used in investigating the anti-obesity effects of an aqueous extract and isoquercitrin from Apocynum venetum L. The aqueous extract and the signal molecule isoquercitrin significantly reduced the body weight gain, food intake, water consumption, and fasting blood glucose, plasma triglyceride and total cholesterol levels of the obese mice. Furthermore, the mechanism of action of isoquercitrin was explored through RT-PCR analyses and uptake experiments of adenosine 5'-monophosphate-activated protein kinase (AMPK) and sterol regulatory-element binding protein (SREBP-1c) inhibitors and glucose. The indexes of SREBP-1c, fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD), and cluster of differentiation 36 (CD36) in obese mice significantly increased but returned to normal levels after the administration of isoquercitrin. Meanwhile, the anti-obesity effect of isoquercitrin was diminished by the inhibitors of AMPK and SREBP-1c. In addition, intestinal glucose uptake in normal mice was significantly inhibited after the oral administration of isoquercitrin. Moreover, 2D gel electrophoresis based proteome-wide cellular thermal shift assay (CETSA) showed that the potential target proteins of isoquercitrin were C-1-tetrahydrofolate synthase, carbonyl reductase, and glutathione S-transferase P. These results suggested that isoquercitrin produces an anti-obesity effect by targeting the above-mentioned proteins and regulating the AMPK/SREBP-1c signaling pathway and potentially prevents obesity and obesity-related metabolic disorders.