PIK3CA encodes the p110α catalytic subunit of PI3K and is frequently mutated in human cancers, including ∼30% of colorectal cancer. Oncogenic mutations in PIK3CA render colorectal cancers more dependent on glutamine. Here we report that the glutaminase inhibitor CB-839 preferentially inhibits xenograft growth of PIK3CA-mutant, but not wild-type (WT), colorectal cancers. Moreover, the combination of CB-839 and 5-fluorouracil (5-FU) induces PIK3CA-mutant tumor regression in xenograft models. CB-839 treatment increased reactive oxygen species and caused nuclear translocation of Nrf2, which in turn upregulated mRNA expression of uridine phosphorylase 1 (UPP1). UPP1 facilitated the conversion of 5-FU to its active compound, thereby enhancing the inhibition of thymidylate synthase. Consistently, knockout of UPP1 abrogated the tumor inhibitory effect of combined CB-839 and 5-FU administration. A phase I clinical trial showed that the combination of CB-839 and capecitabine, a prodrug of 5-FU, was well tolerated at biologically-active doses. Although not designed to test efficacy, an exploratory analysis of the phase I data showed a trend that PIK3CA-mutant patients with colorectal cancer might derive greater benefit from this treatment strategy as compared with PIK3CA WT patients with colorectal cancer. These results effectively demonstrate that targeting glutamine metabolism may be an effective approach for treating patients with PIK3CA-mutant colorectal cancers and warrants further clinical evaluation. Significance: Preclinical and clinical trial data suggest that the combination of CB-839 with capecitabine could serve as an effective treatment for PIK3CA-mutant colorectal cancers.

A liquid chromatography/mass spectrometry (LC-MS) method has been developed and validated for the determination of the anticancer agent gemcitabine (dFdC) and its metabolite 2′,2′-difluoro-2′-deoxyuridine (dFdU) in human plasma. An Oasis^® HLB solid phase extraction cartridge was used for plasma sample preparation. Separation of the analytes was achieved with a YMC ODS-AQ (5 μm, 120 Å, <!--ja:math--> mm) column. The initial composition of the mobile phase was 2% methanol/98% 5 mM ammonium acetate at pH 6.8 (v/v), and the flow rate was 0.2 ml/min. An isocratic gradient was used for 3 min, followed by a linear gradient over 4 min to 30% methanol/70% 5 mM ammonium acetate at pH 6.8. The gradient returned to the initial conditions over 2 min and remained there for 6 min. The retention times of dFdC, dFdU, and the internal standard 5′-deoxy-5-fluorouridine (5′-DFUR) were 11.46, 12.63, and 13.58 min. The mass spectrometer was operated under negative electrospray ionization conditions. Single-ion-monitoring (SIM) mode was used for analyte quantitation at m/z 262 for [dFdC–H]⁻, m/z 263 for [dFdU–H]⁻, and m/z 245 for [5′-DFUR–H]⁻. The average recoveries for dFdC, dFdU, and 5′-DFUR were 88.4, 84.6, and 99.3%, respectively. The linear calibration ranges were 5–1000 ng/ml for dFdC, and 5–5000 ng/ml for dFdU. The intra- and inter-assay precisions (%CV) were ≤3 and ≤7% at three concentration levels (50.0, 500, and 5000 ng/ml). The limits of quantitation (defined as 10 times of signal-to-noise ratio) were 3.16 ng/ml for dFdC, and 1.35 ng/ml for dFdU with 50-μl sample injections. This method has been used for measuring plasma concentrations of dFdC and dFdU in samples from adult cancer patients in a Phase I trial of weekly dFdC given as 150 (or lower) mg/(m² 24-h) infusion. The average plasma dFdC concentrations at 22- and 23-h into the infusion were 18.3 and 16.8 ng/ml at 150 and 100 mg/m², respectively; the values for dFdU averaged 2950 and 1372 ng/ml.

Inhibition of uracil DNA glycosylase sensitizes cancer cells to 5-fluorodeoxyuridine through replication fork collapse-induced DNA damage

This work evaluated the use of an alkaline phosphatase plug, slowly migrating in a polyacrylamide-coated capillary filled with buffer/soluble polymer, to convert p-nitrophenylphosphate (which was injected into the capillary as a separate plug, and migrated faster than the enzyme) to p-nitrophenol under a constant applied potential. The elution of assay components was monitored on-capillary at 230 nm. The initial reaction velocity of the enzymecatalyzed reaction was estimated from the peak area ratio of the enzyme product to the internal standard. Using the Lineweaver-Burk plots, an average Michaelis constant (K_M) for alkaline phosphatase was calculated to be 4.8 ± 0.3 mM (n = 4, CV = 6%). With the constant potential electrophoresis (8 kV/57 cm), the method had a detection limit of 4.4 × 10⁻⁵ IU for alkaline phosphatase and a linear range up to 2.1 × 10⁻³ IU.