Evaluation of Glycolytic Response to Multiple Classes of Anti-glioblastoma Drugs by Noninvasive Measurement of Pyruvate Kinase M2 Using [18F]DASA-23

Abstract

Purpose: Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, the key process of tumor metabolism. PKM2 is found in high levels in glioblastoma (GBM) cells with marginal expression within healthy brain tissue, rendering it a key biomarker of GBM metabolic re-programming. Our group has reported the development of a novel radiotracer, 1-((2-fluoro- 6-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA- 23), to non-invasively detect PKM2 levels with positron emission tomography (PET).

Procedure: U87 human GBM cells were treated with the IC50 concentration of various agents used in the treatment of GBM, including alkylating agents (temozolomide, carmustine, lomustine, procarbazine), inhibitor of topoisomerase I (irinotecan), vascular endothelial and epidermal growth factor receptor inhibitors (cediranib and erlotinib, respectively) anti-metabolite (5-fluorouracil), microtubule inhibitor (vincristine), and metabolic agents (dichloroacetate and IDH1 inhibitor ivosidenib). Following drug exposure for three or 6 days (n = 6 replicates per condition), the radiotracer uptake of [18F]DASA-23 and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) was assessed. Changes in PKM2 protein levels were determined via Western blot and correlated to radiotracer uptake.

Results: Significant interactions were found between the treatment agent (n = 12 conditions total comprised 11 drugs and vehicle) and the duration of treatment (3- or 6-day exposure to each drug) on the cellular uptake of [18F]DASA-23 (p = 0.0001). The greatest change in the cellular uptake of [18F]DASA-23 was found after exposure to alkylating agents (p < 0. 0001) followed by irinotecan (p = 0. 0012), erlotinib (p = 0. 02), and 5-fluorouracil (p = 0. 005). Correlation of PKM2 protein levels and [18F]DASA-23 cellular uptake revealed a moderate correlation (r = 0.44, p = 0.15).

Conclusions: These proof of principle studies emphasize the superiority of [18F]DASA-23 to [18F]FDG in detecting the glycolytic response of GBM to multiple classes of anti-neoplastic drugs in cell culture. A clinical trial evaluating the diagnostic utility of [18F]DASA-23 PET in GBM patients (NCT03539731) is ongoing.

Keywords: Glioblastoma; Glycolysis; Pyruvate kinase M2; [18F]DASA-23; [18F]FDG.

Conflict of interest statement

Conflict of Interest

The authors received funding from the following sources: Ben and Catherine Ivy Foundation (Gambhir), American Brain Tumor Association Basic Research Fellowship supported by the Ryan J. Hanrahan Memorial (Patel), Stanford Cancer Institute Fellowship for Cancer Research (Patel), Stanford-Asia Medical Fund C.J. Huang Medical Fellowship (Xie), Stanford School of Medicine Translational Research and Applied Medicine Fellowship (Beinat). The authors report no conflicts of interest.

Figures

Fig. 1.

Pyruvate kinase (PK) catalyzes the final step of glycolysis, resulting in net ATP synthesis through dephosphorylation of PEP. The PKM2 isoform predominates in tumor cells. Two quaternary PKM2 conformations exist as homo-dimeric or - tetrameric forms (shown as two and four blue spheres, respectively). Dimeric PKM2 has a reduced affinity for PEP in comparison to the tetramer, with tumor PKM2 mainly present in the dimeric form. Reduced conversion of PEP to pyruvate allows buildup of glycolytic precursors for the biosynthesis of macromolecules. Tetrameric PKM2 predominates in non-tumor cells. [18F]DASA-23 is a PKM2 activator, reported to bind a pocket at the dimeric PKM2 subunit interface that promotes association of PKM2 subunits into stable tetramers, due to the conformational change associated with tetramer formation, the binding pocket is not available on constituently active tetramers.

Fig. 2.

[18F]DASA-23 and [18F]FDG cellular uptake in untreated cells and cells treated with alkylating agents temozolomide, procarbazine, carmustine, and lomustine. a Cellular uptake of [18F]DASA-23 and [18F]FDG in U87-untreated cells, ****p < 0.0001. b Three-day treatment; untreated vs temozolomide, ***p = 0.0004; untreated vs carmustine, ****p < 0.0001; untreated vs lomustine, **p = 0.002; untreated vs procarbazine, ****p < 0.0001; n = 6 per condition, p values obtained from one-way ANOVA with Bonferroni correction for multiple comparisons. # = significant interaction on [18F]FDG uptake between the alkylating agents and duration of treatment, two-way ANOVA, 6-day data is shown in Fig. 2b. c Six-day treatment; untreated vs temozolomide, ****p < 0.0001; untreated vs carmustine, ****i < 0.0001; untreated vs lomustine, ****p < 0.0001; untreated vs procarbazine, ****p < 0.0001; n = 6 per condition, p values obtained from one-way ANOVA with Bonferroni correction for multiple comparisons. # = significant interaction on [18F]FDG cellular uptake between the alkylating agent and length of treatment duration, two-way ANOVA, 3-day data is shown in Fig. 2a. Combined 3- and 6-day data is shown for [18F]DASA-23 and [18F]FDG in Suppl. Fig. 4a and 5a, respectively. d Percentage reduction of [18F]DASA-23 and [18F]FDG radiotracer uptake relative to untreated cells in the presence of alkylating agents for 3-day treatment, carmustine **p = 0.007, procarbazine *p = 0.03. e Percentage reduction of [18F]DASA-23 and [18F]FDG radiotracer uptake relative to untreated cells in the presence of alkylating agents for 6-day treatment, temozolomide ****p < 0.0001, carmustine ****p < 0.0001, lomustine ****p < 0.0001, procarbazine **p = 0.0007.

Fig. 3.

[18F]DASA-23 and [18F]FDG uptake in untreated cells and cells treated with metabolic agents dichloroacetate and ivosidenib. a Three-day drug treatment; untreated vs dichloroacetate, **p = 0.004; untreated vs ivosidenib, ***p = 0.0004; n = 6 per condition, p values obtained from one-way ANOVA with Bonferroni correction for multiple comparisons. # = significant interaction in [18F]FDG uptake between the metabolic agents and duration of treatment, two-way ANOVA, 6-day data is shown in Fig. 3b. b Six-day treatment; untreated vs dichloroacetate, ****p < 0.0001; untreated vs ivosidenib, ***p = 0.0005; n = 6 per condition, p values obtained from one-way ANOVA with Bonferroni correction for multiple comparisons. # = significant interaction on [18F]FDG cellular uptake between the alkylating agent and length of treatment duration, two-way ANOVA, 3-day data is shown in Fig. 3a. Combined 3- and 6-day data is shown for [18F]DASA-23 and [18F]FDG in Suppl. Fig. 4b and 5b, respectively. C percentage reduction of [18F]DASA-23 and [18F]FDG radiotracer uptake relative to untreated cells in the presence of metabolic agents is shown for 3-day treatment, ivesodinib ***p = 0.0001. d Percentage reduction of [18F]DASA-23 and [18F]FDG radiotracer uptake relative to untreated cells in the presence of metabolic agents is shown for 6-day treatment, dichloroacetate **p = 0.001.

Fig. 4.

[18F]DASA-23 and [18F]FDG cellular uptake in untreated cells and cells treated with the remaining five classes of drugs: cediranib, irinotecan, erlotinib, vincristine, and 5-fluorouracil. a Three-day treatment; untreated vs cediranib, ****p < 0.0001; untreated vs irinotecan, ***p = 0.0005; untreated vs erlotinib, *p = 0.011; untreated vs vincristine, ****p < 0.0001; untreated vs 5-fluorouracil, ****p < 0.0001; n = 6 per condition, p values obtained from one-way ANOVA with Bonferroni correction for multiple comparisons. # = significant interaction in [18F]FDG cellular uptake between the five drug classes and duration of treatment, two-way ANOVA, 6-day data is shown in Fig. 4b. b Six-day treatment; [18F]DASA-23 uptake: untreated vs cediranib, ****p < 0.0001; untreated vs irinotecan, ****p < 0.0001; untreated vs erlotinib, ****p < 0.0001; untreated vs vincristine, ****p < 0.0001; untreated vs 5-fluorouracil, ****p < 0.0001; n = 6 per condition, p values obtained from one-way ANOVA with Bonferroni correction for multiple comparisons. # = significant interaction on [18F]FDG cellular uptake between the alkylating agent and length of treatment duration, two-way ANOVA, 3-day data is shown in Fig. 5a. Combined 3- and 6-day data is shown for [18F]DASA-23 and [18F]FDG in Suppl. Fig. 4c and 5c, respectively. c Percentage reduction of [18F]DASA-23 and [18F]FDG radiotracer uptake relative to untreated cells in the presence of remaining agents is shown for 3-day treatment, cediranib **p = 0.007, erlotinib **p = 0.002, 5-fluorouracil ***p = 0.002. d Percentage reduction of [18F]DASA-23 and [18F]FDG radiotracer uptake relative to untreated cells in the presence of remaining agents is shown for 6-day treatment, cediranib ***p = 0.0003, irinotecan ****p < 0.0001, erlotinib ****p < 0.0001, 5-fluorouracil ****p < 0.0001.

Fig. 5.

Correlation between [18F]DASA-23 cellular uptake and PKM2 protein expression. a Six-day cellular uptake values from each drug condition (n = 11 total) and untreated cells are shown on the y-axis and PKM2/GAPDH ratios obtained from Western blot studies are shown on the x-axis. Values shown are mean ± SEM in both y- and x-directions. b [18F]DASA-23 cellular uptake values for each drug exposure were normalized to those from untreated cells and plotted on the y-axis as normalized [18F]DASA-23 cellular uptake. PKM2/GAPDH ratios for each drug exposure were also normalized to untreated cells and plotted along the x-axis as normalized PKM2/GAPDH. Values shown are mean ± SEM in both y- and x-directions. Pearson’s correlation, r = 0.44, p = 0.15. Dotted lines represent 95 % confidence interval.