A ratiometric fluorescent sensor that reports the ATP/ADP concentration ratio in living cells was created by fusing the bacterial regulatory protein GlnK1 to a circularly permuted fluorescent protein. The sensor detected inhibition of cellular metabolism caused by transient removal of glucose from the cellular medium or administration of a glycolytic inhibitor. We constructed a fluorescent sensor of adenylate nucleotides by combining a circularly permuted variant of GFP with a bacterial regulatory protein, GlnK1, from Methanococcus jannaschii. The sensor's affinity for Mg-ATP was <100 nM, as seen for other members of the bacterial PII regulator family, a surprisingly high affinity given that normal intracellular ATP concentration is in the millimolar range. ADP bound the same site of the sensor as Mg-ATP, competing with it, but produced a smaller change in fluorescence. At physiological ATP and ADP concentrations, the binding site is saturated, but competition between the two substrates causes the sensor to behave as a nearly ideal reporter of the ATP:ADP concentration ratio. This principle for sensing the ratio of two analytes by competition at a high-affinity site probably underlies the normal functioning of PII regulatory proteins. The engineered sensor, Perceval, can be used to monitor the ATP:ADP ratio during live-cell imaging.

Aerobic glycolysis or the Warburg Effect (WE) is characterized by the increased metabolism of glucose to lactate. It remains unknown what quantitative changes to the activity of metabolism are necessary and sufficient for this phenotype. We developed a computational model of glycolysis and an integrated analysis using metabolic control analysis (MCA), metabolomics data, and statistical simulations. We identified and confirmed a novel mode of regulation specific to aerobic glycolysis where flux through GAPDH, the enzyme separating lower and upper glycolysis, is the rate-limiting step in the pathway and the levels of fructose (1,6) bisphosphate (FBP), are predictive of the rate and control points in glycolysis. Strikingly, negative flux control was found and confirmed for several steps thought to be rate-limiting in glycolysis. Together, these findings enumerate the biochemical determinants of the WE and suggest strategies for identifying the contexts in which agents that target glycolysis might be most effective.

Small-cell lung cancer (SCLC) is an aggressive malignancy in which inhibitors of PARP have modest single-agent activity. We performed a phase I/II trial of combination olaparib tablets and temozolomide (OT) in patients with previously treated SCLC. We established a recommended phase II dose of olaparib 200 mg orally twice daily with temozolomide 75 mg/m² daily, both on days 1 to 7 of a 21-day cycle, and expanded to a total of 50 patients. The confirmed overall response rate was 41.7% (20/48 evaluable); median progression-free survival was 4.2 months [95% confidence interval (CI), 2.8–5.7]; and median overall survival was 8.5 months (95% CI, 5.1–11.3). Patient-derived xenografts (PDX) from trial patients recapitulated clinical OT responses, enabling a 32-PDX coclinical trial. This revealed a correlation between low basal expression of inflammatory-response genes and cross-resistance to both OT and standard first-line chemotherapy (etoposide/platinum). These results demonstrate a promising new therapeutic strategy in SCLC and uncover a molecular signature of those tumors most likely to respond. Significance: We demonstrate substantial clinical activity of combination olaparib/temozolomide in relapsed SCLC, revealing a promising new therapeutic strategy for this highly recalcitrant malignancy. Through an integrated coclinical trial in PDXs, we then identify a molecular signature predictive of response to OT, and describe the common molecular features of cross-resistant SCLC. See related commentary by Pacheco and Byers, p. 1340. This article is highlighted in the In This Issue feature, p. 1325