PKM2 activation sensitizes cancer cells to growth inhibition by 2-deoxy-D-glucose

Abstract

Cancer metabolism has emerged as an increasingly attractive target for interfering with tumor growth. Small molecule activators of pyruvate kinase isozyme M2 (PKM2) suppress tumor formation but have an unknown effect on established tumors. We demonstrate that TEPP-46, a PKM2 activator, results in increased glucose consumption, providing the rationale for combining PKM2 activators with the toxic glucose analog, 2-deoxy-D-glucose (2-DG). Combination treatment resulted in reduced viability of a range of cell lines in standard cell culture conditions at concentrations of drugs that had no effect when used alone. This effect was replicated in vivo on established subcutaneous tumors. We further demonstrated the ability to detect acute metabolic differences in combination treatment using hyperpolarized magnetic resonance spectroscopy (MRS). Combination treated tumors displayed a higher pyruvate to lactate ¹³C-label exchange 2 hr post-treatment. This ability to assess the effect of drugs non-invasively may accelerate the implementation and clinical translation of drugs that target cancer metabolism.

Keywords: PKM2; cancer metabolism; hyperpolarized MRI; metabolic imaging; molecular imaging.

Figure 1

(A) Glucose consumption in TEPP46-treated H1299 lung cancer cells significantly increases at 48 hr compared to vehicle-treated cells (1.6 ± 0.6 mM vs. 3.6 ± 0.4 mM, p < 0.05) and (B) a concomitant increase of lactate secretion into culture media that is significantly different after 24 hr in TEPP46-treated cells compared to vehicle (11.8 ± 0.9 mM vs. 9.1± 0.6 mM, p < 0.05). This trend continues 48 hr (18.9 ± 0.6 vs. 13.1 ± 0.8 mM, p < 0.01) that results (C) a change in the color of phenol red in TEPP-46 treated cells (left panel) that was quantified by absorbance at 516 nm with a significant change in PKM2 activated cells (right panel). (D) These changes in metabolism did not result in significantly different changes in viability as measured by MTS oxidation at 24 hr of TEPP46-treated vs DMSO-treated cells (111.1 ± 6.4 vs. 110.3 ± 10.1, p > 0.05) 48 hr (104.5 ± 2.9 vs 97.5 ± 3.2, p > 0.05) or 72 hr (101.1 ± 0.8 vs 102.2 ± 3.7, p > 0.05). All measurements are quoted as averages ± standard deviation of n =3 independent experiments. Statistical significance was determined using student’s t-test, with p < 0.05 deemed significant (^*) and p < 0.01 (^**).

Figure 2

(A) Viability of a panel of cell lines (3 breast and 2 lung cancer) assessed by MTT assay after treatment using either TEPP-46, 2-DG or a combination of both. (B) Colony forming assay performed on H1299 lung cancer cells seeded at low density in the presence of monotherapy or combination therapy with TEPP-46 and 2-DG. (C) Representative images from a scratch-wound assay 48 h after application of either DMSO, TEPP-46, 2-DG or combination treatment. H1299 cells were grown to confluency and a single scratch was introduced to the monolayer before treatments were applied. Metabolic profiling of cells treated with drugs reveal perturbations in glycolytic intermediates, with combination therapy resulting in the lowest levels of glucose (D), glucose-6-phosphate (E) and fructose-1,6-bisphosphate (F) in cell extracts.

Figure 3

(A) Kaplan-Meier curve of four cohorts of mice (n = 5 each) treated with DMSO control, mono-, or combination therapy of TEPP-46 and 2-DG. Treatment was commenced after successful engraftment of xenograft (3-mm caliper measurement, approximately 10 days post-inoculation) and study was terminated as soon as tumors reached terminal diameters as defined by institutional guidelines. All treatments resulted in no significant difference in body weight at sacrifice compared to DMSO control (B). Metabolite profiling revealed higher levels of saturated fatty acids palmitate (C), stearate (D) and arachidate (E) as well as unsaturated fatty acids 5-dodecenoate (F) and adrenate (G) in tumors that continued growing 3 months post-combination treatment as compared to tumors that were combination-treated for 1 month.

Figure 4. Acute metabolic responses of TEPP-46 and 2-DG combination treatment in subcutaneous tumor measured by hyperpolarized ¹³C MR spectroscopic imaging

Dynamic change in pyruvate metabolism was imaged over tumor-bearing slice (A, B) every 3 s. Time-averaged ¹³C-pyruvate, ¹³C-lactate, and lactate-to-pyruvate ratio maps (C, D) as well as dynamic images (E, F) displayed increased lactate labeling from the injected ¹³C-pyruvate in tumors from a representative combination treated mouse as compared to pre-treatment (G, H). No significant change in the lactate-to-pyruvate ratio was observed in DMSO-treated group (I).