Elevated glucose consumption is fundamental to cancer, but selectively targeting this pathway is challenging. We develop a high-throughput assay for measuring glucose consumption and use it to screen non-small-cell lung cancer cell lines against bioactive small molecules. We identify Milciclib that blocks glucose consumption in H460 and H1975, but not in HCC827 or A549 cells, by decreasing SLC2A1 (GLUT1) mRNA and protein levels and by inhibiting glucose transport. Milciclib blocks glucose consumption by targeting cyclin-dependent kinase 7 (CDK7) similar to other CDK7 inhibitors including THZ1 and LDC4297. Enhanced PIK3CA signaling leads to CDK7 phosphorylation, which promotes RNA Polymerase II phosphorylation and transcription. Milciclib, THZ1, and LDC4297 lead to a reduction in RNA Polymerase II phosphorylation on the SLC2A1 promoter. These data indicate that our high-throughput assay can identify compounds that regulate glucose consumption and that CDK7 is a key regulator of glucose consumption in cells with an activated PI3K pathway.

Background: Conventional scale production of small batches of PET tracers (e.g. for preclinical imaging) is an inefficient use of resources. Using O-(2-[ 18 F]fluoroethyl)-Ltyrosine ([ 18 F]FET), we demonstrate that simple microvolume radiosynthesis techniques can improve the efficiency of production by consuming tiny amounts of precursor, and maintaining high molar activity of the tracers even with low starting activity. Procedures: The synthesis was carried out in microvolume droplets manipulated on a disposable patterned silicon "chip" affixed to a heater. A droplet of [ 18 F]fluoride containing TBAHCO 3 was first deposited onto a chip and dried at 100°C. Subsequently, a droplet containing 60 nmol of precursor was added to the chip and the fluorination reaction was performed at 90°C for 5 min. Removal of protecting groups was accomplished with a droplet of HCl heated at 90°C for 3 min. Finally, the crude product was collected in a methanol-water mixture, purified via analytical-scale radio-HPLC and formulated in saline. As a demonstration, using [ 18 F]FET produced on the chip, we prepared aliquots with different molar activities to explore the impact on preclinical PET imaging of tumor-bearing mice. Results: The microdroplet synthesis exhibited an overall decay-corrected radiochemical yield of 55 ± 7% (n = 4) after purification and formulation. When automated, the synthesis could be completed in 35 min. Starting with < 370 MBq of activity,~150 MBq of [ 18 F]FET could be produced, sufficient for multiple in vivo experiments, with high molar activities (48-119 GBq/μmol). The demonstration imaging study revealed the uptake of [ 18 F]FET in subcutaneous tumors, but no significant differences in tumor uptake as a result of molar activity differences (ranging 0.37-48 GBq/μmol) were observed. Conclusions: A microdroplet synthesis of [ 18 F]FET was developed demonstrating low reagent consumption, high yield, and high molar activity. The approach can be expanded to tracers other than [ 18 F]FET, and adapted to produce higher quantities of the tracer sufficient for clinical PET imaging.

The mammalian heart switches its main metabolic substrate from glucose to fatty acids shortly after birth. This metabolic switch coincides with the loss of regenerative capacity in the heart. However, it is unknown whether glucose metabolism regulates heart regeneration. Here, we report that glucose metabolism is a determinant of regenerative capacity in the neonatal mammalian heart. Cardiac-specific overexpression of Glut1, the embryonic form of constitutively active glucose transporter, resulted in an increase in glucose uptake and concomitant accumulation of glycogen storage in postnatal heart. Upon cryoinjury, Glut1 transgenic hearts showed higher regenerative capacity with less fibrosis than non-transgenic control hearts. Interestingly, flow cytometry analysis revealed two distinct populations of ventricular cardiomyocytes: Tnnt2-high and Tnnt2-low cardiomyocytes, the latter of which showed significantly higher mitotic activity in response to high intracellular glucose in Glut1 transgenic hearts. Metabolic profiling shows that Glut1-transgenic hearts have a significant increase in the glucose metabolites including nucleotides upon injury. Inhibition of the nucleotide biosynthesis abrogated the regenerative advantage of high intra-cardiomyocyte glucose level, suggesting that the glucose enhances the cardiomyocyte regeneration through the supply of nucleotides. Our data suggest that the increase in glucose metabolism promotes cardiac regeneration in neonatal mouse heart.