A Clinical PET Imaging Tracer ([18F]DASA-23) to Monitor Pyruvate Kinase M2-Induced Glycolytic Reprogramming in Glioblastoma

Abstract

Purpose: Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, a key process of cancer metabolism. PKM2 is preferentially expressed by glioblastoma (GBM) cells with minimal expression in healthy brain. We describe the development, validation, and translation of a novel PET tracer to study PKM2 in GBM. We evaluated 1-((2-fluoro-6-[¹⁸F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([¹⁸F]DASA-23) in cell culture, mouse models of GBM, healthy human volunteers, and patients with GBM.

Experimental design: [¹⁸F]DASA-23 was synthesized with a molar activity of 100.47 ± 29.58 GBq/μmol and radiochemical purity >95%. We performed initial testing of [¹⁸F]DASA-23 in GBM cell culture and human GBM xenografts implanted orthotopically into mice. Next, we produced [¹⁸F]DASA-23 under FDA oversight, and evaluated it in healthy volunteers and a pilot cohort of patients with glioma.

Results: In mouse imaging studies, [¹⁸F]DASA-23 clearly delineated the U87 GBM from surrounding healthy brain tissue and had a tumor-to-brain ratio of 3.6 ± 0.5. In human volunteers, [¹⁸F]DASA-23 crossed the intact blood-brain barrier and was rapidly cleared. In patients with GBM, [¹⁸F]DASA-23 successfully outlined tumors visible on contrast-enhanced MRI. The uptake of [¹⁸F]DASA-23 was markedly elevated in GBMs compared with normal brain, and it identified a metabolic nonresponder within 1 week of treatment initiation.

Conclusions: We developed and translated [¹⁸F]DASA-23 as a new tracer that demonstrated the visualization of aberrantly expressed PKM2 for the first time in human subjects. These results warrant further clinical evaluation of [¹⁸F]DASA-23 to assess its utility for imaging therapy-induced normalization of aberrant cancer metabolism.

Figure 1.

Initial development of [¹⁸F]DASA-23 as a PKM2 radiopharmaceutical. A Schematic illustrating the impact of PKM2 on tumor cell glycolysis. The PKM2 isoform is found in proliferating non-tumor and tumor cells. PKM1 is found in the healthy brain. Two quaternary PKM2 isoforms exist as homodimeric and homotetrameric forms (represented as orange circles). Dimeric PKM2 has a reduced affinity for phosphoenolpyruvate compared to tetrameric PKM2. Dimeric PKM2 predominates in tumor cells. A reduced conversion of phosphoenolpyruvate leads to a diminished production of pyruvate and allows the upstream accumulation of glycolytic intermediates that can support the biosynthesis of cellular macromolecules and production of new daughter cells. In contrast, PKM1 is found in the healthy brain and has high constitutive activity for phosphoenolpyruvate. B Cellular uptake and retention profile of [¹⁸F]DASA-23 over time in a human glioma cell panel including LN18 (IDH1 wild-type, MGMT promoter unmethylated) U87 (IDH1 wild-type, MGMT promoter methylated), U87 IDH1 R132H (MGMT promoter methylated) and GBM206 (IDH1 wild-type, MGMT promoter methylated) cells. One-way ANOVA demonstrated significant differences in uptake across cell lines at the 30- and 60-min time points. Tukey’s multiple comparisons of 30-min data revealed significant changes between U87 and U87 IDH1m R132H (p=0.04), and U87 and GBM206 cells (p=0.03). Tukey’s multiple comparisons of 60-min data showed significant changes between LN18 and U87 IDH1 R132H (p=0.03,) LN18 and GBM206 (p=0.02), and U87 and GBM206 (p=0.02). C Representative maximum intensity projection (MIP) of [¹⁸F]DASA-23 in a healthy mouse. Image is summed over 60-min. D Time activity curve of [¹⁸F]DASA-23 in the healthy mouse brain showing initial uptake and then rapid clearance. E Time activity curves reflecting [¹⁸F]DASA-23 renal and hepatobiliary clearance.

Figure 2.

Evaluation of [¹⁸F]DASA-23 in orthotopic U87-GFP/luc GBM mice. A Representative images of a mouse bearing an orthotopic U87-GFP/luc GBM (white arrow), the presence of the tumor is confirmed with T2-weighted MRI. A representative [¹⁸F]DASA-23 fused PET/CT/MR image is shown summed 10-30 min post tracer administration. B Time activity curves illustrating the uptake and clearance of [¹⁸F]DASA-23 over time in the U87-GFP/luc GBM and contralateral normal brain, p<0.0001, n=8. C Quantification of [¹⁸F]DASA-23 radioactivity in U87-GFP/luc GBM and contralateral normal brain at 30-min post tracer administration, t-test, p<0.0001, n=8. D Representative [¹⁸F]DASA-23 autoradiography in mice bearing orthotopic U87-GFP/luc GBM at 30-mins post tracer administration. E H&E stain of an adjacent section showing the presence of the highly cellular GBM. F Expression profile of PKM2 in the brain of mice bearing orthotopic U87-GFP/luc GBM using flow cytometry (n=4).

Figure 3.

Evaluation of [¹⁸F]DASA-23 in healthy human volunteers. A Representative [¹⁸F]DASA-23 whole body maximum intensity projections show the biodistribution of the PET tracer at various time points post tracer administration in volunteer 3. B Representative axial [¹⁸F]DASA-23 PET images of a healthy human brain at various summed time points post tracer administration. C Time activity curves showing [¹⁸F]DASA-23 uptake and clearance in the cerebral cortices, D posterior fossa and E white matter in the left (L) and right (R) brain hemispheres.

Figure 4.

Evaluation of serial [¹⁸F]DASA-23 imaging in recurrent GBM patient IC-1. A Representative contrast-enhanced T1-weighted MRI shown in the axial plane. Yellow arrow indicates tumor, while red arrow indicates the posterior medial portion of the tumor that does not show substantial contrast enhancement. B [¹⁸F]FDG PET/CT shown in the axial plane, yellow arrow indicates area of [¹⁸F]FDG uptake corresponding to the area of contrast enhancement. This scan was obtained 9 days prior to the [¹⁸F]DASA-23 PET scan. C Representative pre-therapy summed 30-60 min [¹⁸F]DASA-23 PET, and D Representative post-therapy summed 30-60 min [¹⁸F]DASA-23 PET, yellow arrow indicates tumor, red arrow indicates the posterior medial portion of the tumor that did not show substantial MRI contrast enhancement, but [¹⁸F]DASA-23 uptake is evident. E Representative post-therapy contrast-enhanced T1-weighted MRI shown in the axial plane. F A subsequent standard of care contrast enhanced T1-weighted MRI is shown in the same axial plane and reveals tumor progression within the areas of [¹⁸F]DASA-23 uptake at the medial and lateral posterior portions of the left frontal mass. G [¹⁸F]DASA-23 SUV distribution within the lesion pre- and post-therapy. H&E H and PKM2 IHC I of the resected tumor tissue shown at 10×, scale bar indicates 300 μm. J Non-significant change (t-test) in cellular uptake of [¹⁸F]DASA-23 in TP459 neurospheres derived from patient IC-2 following treatment with the IC₅₀ dose of TMZ. TMZ = temozolomide, tx = treatment, ns = not significant.

Figure 5.

Evaluation of [¹⁸F]DASA-23 in GBM patient IC-2. A Representative contrast-enhanced T1-weighted MRI shown in the coronal plane. B Representative 30-60 min summed [¹⁸F]DASA-23 PET shows high uptake of the radiotracer in the area corresponding to contrast enhancement. C Fused 30-60 min summed [¹⁸F]DASA-23 PET/MRI. Yellow arrows indicate the location of the tumor. H&E staining (D) and PKM2 IHC (E) of the biopsied tissue shown at 0.4×, scale bar indicates 4mm. H&E (F) and PKM2 IHC (G) of the area shown within the black box in the insert confirm the expression of PKM2 within the malignant cells, images are shown at 10× and scale bar represents 300 μm.

Figure 6.

Evaluation of [¹⁸F]DASA-23 in patients IC-3 ((WHO Grade IV diffuse midline glioma, H3 K27M-mutant) and IC-4 (WHO grade III recurrent anaplastic astrocytoma). A Representative contrast-enhanced T1-weighted MRI of IC-3 shown in the axial plane. B Representative 30-60 min summed [¹⁸F]DASA-23 PET shows two focal areas of tracer uptake in IC-3. C Fused 30-60 min summed [¹⁸F]DASA-23 PET/MRI in IC-3. Yellow arrows indicate the location of the tumor. H&E D and PKM2 IHC E of the biopsied tissue confirm the expression of PKM2 within the malignant cells, images are shown at 10× and scale bar represents 300 μm. F Representative contrast-enhanced T1-weighted MRI of IC-4 shown in the axial plane. G Representative 30-60 min summed [¹⁸F]DASA-23 PET shows heterogeneous tracer uptake in IC-4. H Fused 30-60 min summed [¹⁸F]DASA-23 PET/MRI in IC-4. Yellow arrows indicate the location of the tumor. H&E I and PKM2 IHC J of the resected tumor confirm the expression of PKM2 within the malignant cells, images are shown at 10× and scale bar represents 300 μm.