Inhibition of monocarboxyate transporter 1 by AZD3965 as a novel therapeutic approach for diffuse large B-cell lymphoma and Burkitt lymphoma

Abstract

Inhibition of monocarboxylate transporter 1 has been proposed as a therapeutic approach to perturb lactate shuttling in tumor cells that lack monocarboxylate transporter 4. We examined the monocarboxylate transporter 1 inhibitor AZD3965, currently in phase I clinical studies, as a potential therapy for diffuse large B-cell lymphoma and Burkitt lymphoma. Whilst extensive monocarboxylate transporter 1 protein was found in 120 diffuse large B-cell lymphoma and 10 Burkitt lymphoma patients' tumors, monocarboxylate transporter 4 protein expression was undetectable in 73% of the diffuse large B-cell lymphoma samples and undetectable or negligible in each Burkitt lymphoma sample. AZD3965 treatment led to a rapid accumulation of intracellular lactate in a panel of lymphoma cell lines with low monocarboxylate transporter 4 protein expression and potently inhibited their proliferation. Metabolic changes induced by AZD3965 in lymphoma cells were consistent with a feedback inhibition of glycolysis. A profound cytostatic response was also observed in vivo: daily oral AZD3965 treatment for 24 days inhibited CA46 Burkitt lymphoma growth by 99%. Continuous exposure of CA46 cells to AZD3965 for 7 weeks in vitro resulted in a greater dependency upon oxidative phosphorylation. Combining AZD3965 with an inhibitor of mitochondrial complex I (central to oxidative phosphorylation) induced significant lymphoma cell death in vitro and reduced CA46 disease burden in vivo These data support clinical examination of AZD3965 in Burkitt lymphoma and diffuse large B-cell lymphoma patients with low tumor monocarboxylate transporter 4 expression and highlight the potential of combination strategies to optimally target the metabolic phenotype of tumors.

Copyright© 2017 Ferrata Storti Foundation.

Figures

Figure 1.

Diffuse large B-cell lymphoma and Burkitt lymphoma are appropriate diseases for AZD3965 treatment. (A) H-score analysis of MCT1 and MCT4 tumor cell protein expression in 120 samples from DLBCL and 10 BL patient samples. A sample was considered negative (H-score of 0) when no staining was evident on tumor cells, staining on stromal cells or inflammatory infiltrate being excluded from the analysis. Representative MCT1 and MCT4 immunohistochemical staining from two DLBCL samples (i and ii) and one BL sample (iii) are shown. (B) Pie charts indicate that the majority of DLBCL samples are MCT4 negative and the relative proportion of MCT4 negative samples is similar in both activated B-cell (ABC) and germinal center B-cell (GCB) subsets. (C) MCT4 vs. MCT1 H-score plot for the DLBCL samples in relation to ABC/GCB classification.

Figure 2.

MCT1 inhibition induces rapid accumulation of lactate and significant anti-proliferative activity in diffuse large B-cell lymphoma and Burkitt lymphoma cell lines. (A) MCT1 and MCT4 protein expression in cell lines using GAPDH as a loading control. (B) Intracellular lactate in cell lines following 24 h incubation with AZD3965 (1 μM) or vehicle. (C) Concentration and time dependency of intracellular lactate accumulation in CA46 cells following treatment with AZD3965 or vehicle. (D–F) Sensitivity of BL or DLBCL cells treated with AZD3965 for 72 h assessed by XTT assay. (G and H) Cell number and viability following AZD3965 (100 nM) treatment for 72 h. (I) Cell viability following an extended 120 h exposure to AZD3965 (100 nM). Graphs show the means of ≥3 independent experiments ± SEM. *P<0.05, **P<0.01, ***P<0.001 by unpaired two-tailed t-test.

Figure 3.

AZD3965 alters cellular metabolism in vitro and in vivo causing growth inhibition. (A) Levels of tricarboxylic acid (TCA) cycle and glycolytic intermediates in cell lines following 2 h exposure to AZD3965 (100 nM) determined by liquid chromatographymass spectrometry. Significantly altered metabolites (P<0.05) are expressed as log2 fold-change relative to vehicle-treated control. αKG: alphaketoglutarate; FBP: fructose-bisphosphate; F1P: fructose-1-phosphate; F6P: fructose-6-phosphate; GAP: glyceraldehyde-3-phosphate; G1P: glucose-1-phosphate; G6P: glucose-6-phosphate. (B) NSG mice with subcutaneous CA46 xenografts were treated with AZD3965 (100 mg/kg) or vehicle and tumors collected after 2 h. Lactate concentrations were normalized to protein. (C) Significantly altered (unpaired two-tailed t-test) intratumoral metabolite levels determined by gas chromatography-mass spectrometry. (D) NSG mice were inoculated intravenously with luciferase-expressing CA46 cells and 6 days later (treatment day 0) treated with AZD3965 (100 mg/kg, BID) or vehicle for 24 days. Representative images from two mice in the AZD3965 and vehicle-treated groups using different radiance scales (p/sec/cm2/sr) for mice prior to treatment and during treatment to avoid image saturation. (E) Mean total flux from AZD3965 and vehicle-treated mice (n=8 per group). (F) Spleen weights from AZD3965 and vehicle-treated mice. Reference historical spleen weights from NSG mice were 0.02–0.05 g. (G) Immunohistochemical analysis of CA46 infiltration via anti-CD20 staining of bone marrow and spleen sections from mice treated with AZD3965 or vehicle. Statistical significance was assessed by an unpaired two-tailed t-test *P<0.05, ***P<0.001.

Figure 4.

Acquired resistance to AZD3965 in vitro is associated with increased oxidative metabolism. (A) The sensitivity of CA46 and CA46-R cells to AZD3965 (72 h treatment) determined by an XTT assay and cell counting. (B) Intracellular accumulation of lactate determined after 24 h exposure to AZD3965 (1 μM). MCT1, MCT4 and CD147 protein levels assessed by western blotting. (C) Extracellular acidification rate (ECAR) in CA46 and CA46-R with and without treatment with AZD3965 (100 nM) or vehicle. Oxygen consumption rate (OCR) in CA46 and CA46-R cells, indicating the effects following addition of oligomycin, FCCP and antimycin. ECAR and OCR values (mean ± SEM) are normalized to protein expression and representative of three independent experiments.

Figure 5.

Combining AZD3965 with inhibitors of mitochondrial complex I induces death of Burkitt lymphoma cells. Viable cell numbers were determined by cell counting with trypan blue exclusion over a 72 h period, following treatment with AZD3965, a complex I inhibitor, or the combination. (A) Raji cells treated with vehicle, AZD3965 (100 nM), metformin (1 mM) or the combination. (B) Raji cells treated with vehicle, AZD3965 (5 nM), BAY 87-2243 (100 nM) or the combination. (C) CA46 and (D) CA46-R cells treated with vehicle, AZD3965 (10 nM), BAY 87-2243 (10 nM) or the combination. All graphs show the means of ≥3 independent experiments ± SEM.

Figure 6.

Combining AZD3965 with an inhibitor of mitochondrial complex I in vivo. (A) Schema indicating treatment duration and scan intervals. (B) Pre- and post-treatment bioluminescent signals for mice within each group with a representative image from one of the mice that received the combination (inset). (C) Change in signal intensity subsequent to treatment. Graph shows the mean + SD total flux (n ≥5 per group).